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like 1

2020-12-10T09:04:12.207Z

1974-06-01T00:00:00.000Z

237235488

s2

Evaluation of Porcelain Cup Soil Water Samplers for Bacteriological Sampling The validity of obtaining soil water for fecal coliform analyses by porcelain cup soil water samplers was examined. Numbers from samples of manure slurry drawn through porcelain cups were reduced 100- to 10,000,000-fold compared to numbers obtained from the external manure slurry, and 65% of the cups yielded coliform-free samples. Fecal coliforms adsorbed to cups apparently were released, thus influencing the counts of subsequent samples. Fecal coliforms persisted in soil water samplers buried in soil and thus could significantly influence the coliform counts of water samples obtained a month later. These studies indicate that porcelain cup soil water samplers do not yield valid water samples for fecal coliform analyses. The validity of obtaining soil water for fecal coliform analyses by porcelain cup soil water samplers was examined. Numbers from samples of manure slurry drawn through porcelain cups were reduced 100to 10,000,000-fold compared to numbers obtained from the external manure slurry, and 65% of the cups yielded coliform-free samples. Fecal coliforms adsorbed to cups apparently were released, thus influencing the counts of subsequent samples. Fecal coliforms persisted in soil water samplers buried in soil and thus could significantly influence the coliform counts of water samples obtained a month later. These studies indicate that porcelain cup soil water samplers do not yield valid water samples for fecal coliform analyses. The survival and subsequent downward dispersal of fecal organisms applied to soil in raw manure slurry have recently been quantitatively described (2,3). These findings indicated a high potential for groundwater contamination in areas where excessive numbers of fecal bacteria were applied to a well-drained sandy soil having a high water table. During the course of these studies, the use of porcelain cup soil water samplers placed in the soil at various depths to obtain underground water samples for coliform analyses was suggested. These samplers have previously been used to collect water samples for coliform analyses elsewhere (4,5). The purpose of this study was to determine if soil water samplers (1900-A, Soil Moisture Equipment Corp., Santa Barbara, Calif.) could be used to obtain valid water samples for fecal coliform analyses. A sampler consisted of a plastic tube terminating in a round-bottom, porous porcelain cup with a wall thickness of 0.24 cm and a pore size of 3 to 8 ,gm (Fig. 1). Fresh cow manure slurry was obtained from a cement holding tank 30 min before it was to be applied on land as previously described (3). Settleable solids were allowed to separate for 1 h. The suspended solids were decanted and used as the source of fecal coliforms. Twenty soil water samplers were steamed for 10 min at 100 C, cooled to room temperature, and submerged in beakers containing decanted slurry. Ten samplers were connected simultaneously to I Florida Agricultural Experiment Station Journal Series No. 5323. a manifold, and a vacuum of 100 mm was applied to each sampler for 20 min until an approximate 200-ml sample was obtained. The fecal coliform numbers of these samples and the external slurry before applying suction were determined by the three-tube most-probablenumber (MPN) and membrane filter (MF) techniques (1). Fresh slurry was added to the beakers when necessary. After samples were removed from the samplers, the outer walls of the cups were scrubbed with steel wool, rinsed, and submerged in 2-liter beakers containing sterile deionized water. Samples (200 ml) were obtained and treated as before to determine fecal coliform numbers. The cup conductance K20, a measure of the volume of water passing through the cup wall per unit of time per unit of pressure difference (ml/min/atm), was determined for each acidwashed sampler, in triplicate, by the method of Richards (6). The samplers were acid-washed to remove organic matter in the pores. Fifty milliliters of sterile deionized water or fresh slurry containing 7 x 107 fecal coliforms (MPN) was added to each of two steamed and cooled samplers. The four samplers were stoppered and buried with the cups at a 30-cm depth in Arrendondo fine sand (Grossarenic Paleudalf). After 33 days, the samplers were withdrawn and the outer porcelain walls were cleaned with steel wool. The samplers were then submerged in sterile deionized water. A vacuum was applied to obtain 200-ml samples, and the fecal coliform numbers of the samples were determined. The fecal coliform numbers in fresh slurry were 2.4 x 108 cells/100 ml and 1.1 x 107 cells/100 ml as determined by the MPN and MF techniques, respectively. The counts obtained from suction samples are shown in Table 1. None of the fecal coliform counts obtained from suction samples were representative of the counts in the slurry (columns 2 and 3, Table 1). A 100to 10,000,000-fold reduction in numbers of fecal coliforms was observed, depending on the sampler. Fecal coliforms could not be detected in 65% of the samples. In all cases, fecal coliforms detected by the MPN method were also detected by the MF method. The results of a statistical test indicated that most of the MPN values of the samples were significantly lower (at a = 0.01 level) than the corresponding estimated value outside the cups. It is evident that loss of organisms occurred during passage through the cups. The fecal coliform counts of the 200-ml samples obtained after transfer of cups to sterile water are presented in columns 4 and 5 of Table 1. Among the 20 cups, 9 (45%) contained residual coliforms carried over in the water samples. These results indicated that the retention of fecal coliforms in the cups may significantly influence (at a = 0.01 level) the counts of subsequent samples by the apparent desorption of fecal coliforms on or in the cups. Although the water outside the cups was sterile, samples obtained from four samplers yielded sufficient numbers of coliforms (> 1,000 total coliforms/100 ml) to reject the water for use as "class 1 waters" (withdrawn for treatment and distributed as a potable supply) in Florida (7). There appeared to be an equal probability (0.2) that the residual MPN value may be either greater or less than the MPN value from the previous sampling. If there were a constant level of reduction in fecal coliform numbers as the slurry passed through each cup, a simple conversion factor compensating for this constant loss might increase the usefulness of the samplers. On the contrary, the recovery of fecal coliforms (MPN) exhibited large variations from cup to cup. The estimate of the standard deviation (SD) among cups based on 20 samples (column 2, Table 1) was approximately 770 cells/100 ml. This could be an underestimate since the MPN values >2,400 (cups 14 and 18) were taken as 2,400 in calculating the estimate of 770. This SD value was sufficiently large to indicate that a simple conversion factor compensating for loss during suction would be unreliable. The averages of triplicate K20 values for each sampler (column 6, Table 1) indicated no evidence of a correlation between cup conductance and the recovery of fecal coliforms inside the cups. The standard deviation of the K20 value (0.858) was greater than ½/2 of the mean (1.505) for the 20 cups. This wide variation in cup conductance represented an undesirable characteristic of variable water-flow rates from cup to cup. Fecal coliforms retained in the samplers persisted and significantly influenced the coliform counts of water samples obtained 33 days later. Approximately 1% of the viable fecal coliforms applied were recovered after this period (8.6 x 105 cells recovered from 7.0 x 107 cells inoculated). Fecal coliforms were not recovered from uninoculated samplers, so the soil surrounding the cups did not contribute fecal coliforms to the analyses. A 99% reduction in numbers of fecal coliforms in a Scranton fine sand (Mollic Psammaquent) occurred within 14 days (3). The data suggested that mortality of fecal coliforms was more rapid in this soil than in the cups. Apparently, the cups protected the fecal coliforms from the soil environment. Microbiological surveillance should be an important component of all experimental waste water renovation programs. Land application is currently an attractive alternative to the discharge of sewage effluent and animal wastes into surface waters. Results of this study indicated that soil water samplers with porous cups should not be used to obtain soil water samples for fecal coliform analyses.

v3-fos

2020-12-10T09:04:12.824Z

1974-04-01T00:00:00.000Z

237232255

s2

Form of Selenium in Selenite Enrichment Media for Isolation of Salmonellae Selenite-F and selenite-cystine media, commercially available for the routine isolation of salmonellae, were treated by anion exchange chromatography to separate the selenium from other components of the media. A chemical assay, based on an ascorbic acid reduction, showed that the selenium was all in the form of selenite. NaHSeO2 and an undefined mixture of polypeptones; other substances such as cystine, which apparently enhance the selectivity of selenite (6), are sometimes included. A reaction between selenite and low-molecular-weight thiols is well established (1), and a stoichiometric reaction between selenite and the thiol groups of proteins has also been described (2). Smith (10), on the basis of growth studies with these media, concluded that selenite reacts with and becomes bound to other components of the selenite medium and that the resulting seleniumcontaining substances, rather than selenite itself, are the ones that inhibit the growth of sensitive organisms. A direct analysis to determine the exact nature of the selenium, however, has not been reported. This note will present evidence that the selenium in both Selenite-F and selenite-cystine media remains as selenite. Selenite-F and selenite-cystine media were purchased from BioQuest, Cockeysville, Md. Selenite-F medium was prepared without heating, according to the manufacturer's directions. 7"Se-selenious acid was obtained from Union Carbide Corp., Tuxedo, N.Y., and from Amersham/Searle Corp., Arlington Heights, Ill. To remove small amounts of radioactive contaminants, the 75Se-selenious acid was purified by anion exchange chromatography (9). To determine whether the selenium present in the selenite medium was readily precipitable by ascorbic acid, and to determine the amount of such selenium, a selenium assay adapted from an ascorbic acid reduction method (A. A. Tumanov, N. M. Shakhverdi, and Z. I. Glazunova, Chem. Abstr. 67:3731, 1967) was used. To 2-ml samples that contained from 0 to 200 ,ug of selenium were added 2 ml of 2 N HCl followed by 2 ml of a saturated solution of reduced ascorbic acid. After 5 min, each tube was stirred with a Vortex mixer. Transmission was measured either with a Spectronic-20 at 500 nm or with a Klett-Summerson colorimeter with a blue filter (no. 42). The standard curve with NaHSeO, was linear over a range from 0 to 160 ,ug of Se/ml. The selenium recovered from Selenite-F medium equaled the amount specified by the manufacturer. Unheated selenitecystine broth gave identical results. To determine if selenite becomes associated with components of Selenite-F medium, a mixture of 75Se-selenious acid and medium was examined by gel filtration chromatography on a Sephadex G-10 column. Materials that absorbed at 280 nm (A280) were monitored with a Beckman DB-G spectrophotometer. Radioactivity was monitored with a Packard gamma spectrometer. Figure 1 shows that the peak of radioactivity failed to coincide with any of the A280 peaks. Separation of selenite from A280 materials by anion exchange chromatography is illustrated in Fig. 2. Selenite powder was prepared as described above. Much of the A280 material was removed with water; after A280 had reached a minimum, HCl of pH 2.0 was applied, and the remainder of the A28, material was removed. Radioactivity was eluted by HCl of pH 1.5; it emerged at the position of purified 75Se-selenious acid. No radioactivity was detected in any of the other fractions. A small rise in A280 readings consistently preceded the radioactive peak in each experiment. However, no clear-cut peak accompanied the peak of radioactivity (insert, Fig. 2). A ninhydrin reaction carried out on the radioactive fractions was negative. Identical results were obtained with unheated selenite-cystine medium. Storage of both types of medium in the freezer for several months had no effect on the elution pattern. To determine how much selenium was actually present in the radioactive fractions, they were pooled, and samples were analyzed for selenite by the ascorbic acid reduction method. The selenium recovered in the pooled fractions accounted for all of the selenium that had been placed on the ion exchange columns. The ready precipitability by ascorbic acid is one indication that selenium in Selenite-F and selenite-cystine media is still in the form of selenite. Under the acid conditions of this assay, organic trisulfides (R-S-Se-S-R), known to form when sulfhydryl-containing compounds are treated with selenite, would probably be stable (1,2). The absence of radioactive peaks in 815 FIG. 2. Ag-i-Cl chromatography of Selenite-F medium. Two milliliters of a solution containing 183.7 mg of the medium (x4 the normal concentration) and 75Se-selenious acid as marker was applied to a column of Ag-1-x8, 200-400 mesh, Cl-form, 1 by 56 cm. The column was pretreated with 1 N HCI followed by glass-distilled water until free of A28, materials and until a pH of about 4 to 5 was reached. Two-milliliter fractions were collected. association with A28, peaks in the Sephadex G-10 and anion exchange eluates, as well as the quantitative recoveries of radioactive selenium in the radioactive peaks, show a failure of selenite to react with components of the media. The anion exchange chromatographic separation of selenite from A280 materials and the quantitative recovery of total selenium in the resolved radioactive peaks support this conclusion. Any explanation for the unusual tolerance shown by salmonellae, therefore, must be sought, not in some other form of selenium as has been claimed (10), but in the biochemical properties of selenite.

v3-fos

2017-10-28T00:36:58.831Z

1974-01-01T00:00:00.000Z

32395099

s2

Rapid Methods for Biochemical Testing of Anaerobic Bacteria Rapid biochemical tests for nitrate, indole, gelatin, starch, esculin, and o-nitrophenyl-,B-D-galactopyranoside were performed on 112 strains of anaerobic bacteria. All tests were incubated under aerobic conditions, and results were recorded within 4 h. The tests for nitrate, indole, and starch showed a 95% or greater correlation when compared to the standard biochemical tests. Tests for esculin and gelatin showed an agreement of 86 and 77%, respectively. PathoTec test strips for nitrate, indole, esculin, o-nitrophenyl-,B-D-galactopyranoside, Voges-Proskauer, and urease were also tested and showed encouraging results. The development of simple, rapid, and sensitive biochemical tests for use with the aerobic and facultative bacteria has greatly influenced the speed with which these bacteria may be identified. The main emphasis in the past has been directed toward those bacteria belonging to the family Enterobacteriaceae, as evidenced by the many kits which have become available commercially during the past few years (1,2,9,10,13,14,16). The present study was carried out to determine the feasibility of using rapid biochemical tests for the identification of anaerobic bacteria. MATERIALS AND METHODS All anaerobic bacteria used in the study were recent laboratory isolates saved in chopped meat broth at room temperature in the dark. All organisms had been previously identified by the Virginia Polytechnic Institute (VPI) methodology (6). There were 112 organisms used in the study consisting of 1 Arachnia, 10 Bacteroides fragilis, 10 Bacteroides species, 10 Bifidobacterium, 10 Clostridium perfringens, 10 Clostridium species, 10 Eubacterium, 6 Fusobacterium, 5 Lactobacillus, 10 Peptococcus, 10 Peptostreptococcus, 10 Propionibacterium, and 10 Veillonella. All prereduced media were purchased from Scott Laboratories (Fiskeville, R.I.) and were inoculated using the VPI anaerobic culture system (Bellco). Prior to testing, each strain was subcultured to a fresh blood agar plate (BAP) prepared with Trypticase soy agar, 5% sheep blood, and 1% hemin-vitamin K solution (Scott Laboratories). The plates were incubated anaerobically in a vented GasPak jar evacuated and filled three times with 90% CO, and 10% H.. If the culture was pure, a single well-isolated colony was picked and transferred to prereduced peptone yeast glucose (PYG) broth or chopped meat glucose broth. All further subculturing was carried out from this I Present address: Curriculum in Medical Laboratory Sciences, University of Illinois Medical Center, Chicago, Ill. 60612. broth. In the case of the rapid tests, a fresh BAP supplemented with hemin and vitamin K was inoculated and incubated anaerobically for 24 to 48 h and was the source of inoculum for all the rapid tests. In addition, about 10% of the positive tests were retested with inoculum taken from prereduced supplemented brain heart infusion agar (BHIA) plates with 5% sheep blood added, a roll streak tube, and a PYG broth culture which was 24 to 48 h old. The standard tests used followed the procedures recommended by VPI (6) and were inoculated with 4 drops of an actively growing culture from either PYG or chopped meat glucose broths. The rapid biochemical tests used were: nitrate reduction, indole production, hydrolysis of gelatin, esculin, and starch, and the ONPG test for measuring hydrolysis of o-nitrophenyl-,B-D-galactopyranoside (ONPG) by beta galactosidase. All media were dispensed into tubes (13 by 100 mm). The nitrate test was prepared by dissolving 0.9 g of nitrate broth (Difco) in 100 ml of distilled water. The broth was dispensed in 0.5-ml portions into borosilicate tubes and autoclaved. The tubes were stored at 4 C no longer than 2 weeks. Tryptone broth was prepared by dissolving 1.0 g of tryptone broth (Difco) in 100 ml of distilled water and dispensing in 0.5-ml portions. The tubes were autoclaved and frozen at -20 C until used. The starch substrate was prepared by suspending 0.05 g of soluble starch in 100 ml of physiological saline (0.85% NaCl). The suspension was autoclaved, and the resultant solution was dispensed in 0.5-ml portions and stored at 4 C until used. The ONPG broth was made according to the formula of Cowan and Steel (4), dispensed in 0.5-ml portions, and frozen at -20 C. Esculin hydrolysis was determined by using 0.5 ml of enterococcosel broth (Gibco). The test for gelatin hydrolysis was performed on pieces of undeveloped X-ray film which had been cut into small strips (approximately 0.5 by 5.0 mm). The strips were placed into a 0.5-ml saline suspension of the test organism. All tests were performed with a sterile loop and by scraping up growth from the surface of the agar medium and inoculating directly into the broth substrate. When inoculum was taken SCHRECKENBERGER AND BLAZEVIC from PYG broth, 2 drops of a 24to 48-h culture were pipetted directly into the rapid test medium. All tests were incubated aerobically at 35 C and read within 4 h. Tests for gelatin hydrolysis which were negative after 4 h were reincubated and read after 24 h. Reduction of nitrate was detected by adding 1 drop of reagent A (0.8% sulfanilic acid in 5 N acetic acid) and one drop of reagent B (0.5% alpha-naphthylamine in 5 N acetic acid) to the nitrate broth. The development of a red or pink color was considered positive, whereas the absence of color was considered negative. All negative tests were rechecked by adding a pinch of zinc dust to the nitrate broth. The absence of a red or pink color after the addition of zinc was considered positive. Indole production was detected by adding 2 drops of Kovac reagent to the tryptone broth. A positive reaction was denoted by a deep pink color in the surface layer. Starch hydrolysis was detected by adding 1 drop of a 1:4 dilution of Gram iodine solution (Kopeloff modification) to the starch broth. The development of a deep blue to black color was indicative of no starch hydrolysis. Any change in color as compared to an uninoculated control was considered positive. Esculin hydrolysis was indicated by the development of a brown or black color in the substrate broth. No change in color was considered negative. The gelatin tests were observed for the removal of the green gelatin emulsion from the X-ray strips. In a positive test the gelatin is removed, exposing the blue transparent strip. In a negative test the strip remains green. The ONPG tests were observed for the development of a yellow color, which was considered positive. In a negative reaction the substrate remained colorless. PathoTec strips for nitrate, indole, esculin, urease, Voges-Proskauer (VP), and ONPG were tested with the same 112 organisms. The tests were performed according to the manufacturer's instructions for Enterobacteriaceae, including 4 h of incubation in an aerobic environment. Inoculum was taken from a 24to 48-h anaerobic BAP. About 10% of the tests were performed in triplicate with organisms from BHIA, a roll streak tube, and PYG broth. When the PYG broth was used, 0.3 ml of a 24to 48-h culture was transferred to a test tube (13 by 100 mm), and a PathoTec strip was added. To determine the effect of atmosphere on both the rapid biochemical tests and the PathoTec test strips, about 10% of those organisms giving a positive reaction for a given test were retested simultaneously under aerobic and anaerobic conditions. A double volume of substrate was inoculated with 24to 48-h growth from an anaerobic BAP. The suspension of bacteria and substrate was then divided equally between two test tubes, and one was incubated aerobically at 35 C and the other anaerobically in an evacuation jar (90% CO,, 10% H2) also at 35 C. All media and reagents were checked with positive and negative controls to ensure their accuracy. Table 1 shows the results of the rapid tests compared with the standard tests as performed according to the VPI method. There was a 99% correlation with the indole test. One Clostridium species was positive with the standard test but negative with the rapid test. There was a 97% correlation with the test for starch hydrolysis. One Eubacterium and two Lactobacillus were positive with the standard test and negative with the rapid test. Nitrate gave a 95% correlation; both strains of Bacteroides corrodens, all three strains of Eubacterium lentum, and one Veillonella were positive with the standard method but were negative with the rapid tests. The rapid esculin test showed an 86% correlation with the standard method. There were 14 organisms which were positive with the standard test and negative with the rapid test. These included one Bacteroides fragilis, four Bifidobacterium, one Clostridium perfringens, five Clostridium species, two Fusobacterium, and one Peptostreptococcus. Two Clostridium perfringens were positive with the rapid test but negative with the standard method. Gelatin had the lowest correlation (77%). However, there were 13 instances where the standard test gave a weak positive reaction, and the rapid test was negative. A weak positive reaction in the VPI system is defined as liquification in less than one-half the time required for Table 2 shows the comparison between the PathoTec test strips and the standard method. Indole showed a 96% correlation; three species of Fusobacterium and one Clostridium species were positive with the standard method and negative with the PathoTec strips. Esculin showed an 89% agreement. There were eleven organisms, including four Clostridium species, one Clostridium perfringens, three Bifidobacterium, one Peptostreptococcus, and two Fusobacterium, which were positive with the standard test but negative with PathoTec. One Clostridium perfringens was positive with Pa-thoTec but negative with the standard method. There were 21 organisms which were falsely negative for nitrate reduction by the PathoTec strips. These included two Bacteroides corrodens, two Propionibacterium, four Veillonella, three Eubacterium lentum, and ten Clostridium perfringens. In addition to the indole, esculin, and nitrate strips, PathoTec strips for urease, VP, and ONPG were also tested. The urease and VP strips were negative for most of the organisms tested. One strain of Bacteroides corrodens was urease positive. The ONPG strips compared in every instance with the rapid ONPG tests used routinely in our laboratory. RESULTS Concerning the effect of atmosphere, only the nitrate test seemed to prefer an anaerobic environment. All six organisms which had been falsely negative with the rapid nitrate test when incubated aerobically were positive when the test was incubated anaerobically. Thirteen of the 21 organisms, which were falsely negative for nitrate reduction by the PathoTec strips when incubated aerobically, were positive under anaerobic conditions. Four strains of Clostridium perfringens, two Bacteroides corrodens, and two Eubacterium lentum remained negative even under anaerobic incubation. Atmosphere seemed to have no effect on the tests for esculin hydrolysis. The tests for indole, starch, gelatin, and ONPG all seemed to prefer an aerobic environment as measured by the intensity and speed of the color development. Inoculum taken from the BHIA seemed to work slightly better than inoculum taken from the BAP. However, inoculum taken from the roll streak tube or PYG broth was less satisfactory. DISCUSSION Rapid testing of anaerobic bacteria is not new and has been reported as far back as 1941 by Reed and Orr (12). Clarke and Cowan (3) used several species of Clostridium in their studies of rapid techniques in 1952. Kaufman and Weaver (8) also used several species of Clostridium in their report of a combined media for detection of gelatin hydrolysis and indole production. In 1972, Sutter and Carter (15) evaluated four indole-spot tests for use in anaerobic bacteriology. The results presented here show that rapid testing of anaerobic bacteria is possible, and in most cases can be carried out within 4 h under aerobic conditions. An exception to this rule may be the test for nitrate reductase. Our findings are consistent with others (11,17) who have reported that nitrate reductase seems to have a diminished activity when exposed to oxygen. On the other hand, indole production has been shown to decrease when facultative organisms are incubated anaerobically (7). Fay and Barry (5) found the opposite to be true when testing for indole production with the obligate anaerobes. The discrepancy with our results may be explained by the fact that in on February 1, 2021 by guest http://aem.asm.org/ Downloaded from their system growth was allowed to occur under the anaerobic environment, and thus the increased production of indole may be attributed to an increase in cell mass rather than increased enzyme activity. In our system growth was unlikely, and thus the increased indole production under aerobic conditions can probably be attributed to an increased activity of the enzymes involved. There was a wide range of colors produced with the test for starch hydrolysis. For this reason each test was compared to a negative control. Various colors, from light blue to brown to yellow, were obtained upon addition of the diluted iodine solution. These colors represent various degrees of starch hydrolysis, but, for the purpose of comparison, any color deviation from the negative control was interpreted as a positive reaction. A special precaution might be mentioned concerning the test for gelatin hydrolysis. Prior to reading the test, the tubes containing the gelatin strips should be shaken vigorously to remove any loose gelatin which might be adhering to the strips. Ten of the 21 organisms which showed a false negative reaction with the PathoTec nitrate strips were classified as Clostridium perfringens. The false negatives encountered with these and other species of anaerobic bacteria such as Veillonella, Bacteroides corrodens, and Eubacterium lentum suggest that the sensitivity of these strips might have to be adjusted before the nitrate strips can be recommended for use with the anaerobic bacteria. The effect of inoculum size seems to be consistent with our experience in rapid testing of facultative bacteria. A larger inoculum produces a stronger reaction in a shorter period of time. It is important to note that at all times it is assumed that one is working with a pure culture. Whenever contamination is suspected, a single well-isolated colony must be picked to fresh media to initiate a pure culture. Inoculum taken from a BAP or BHIA generally produced the best results. However, in some instances better growth was achieved on BHIA, and in these instances a stronger reaction was elicited in the rapid test system from this medium. The poorer correlation resulting from the roll tube might be explained by the fact that it was generally difficult to scrape up growth from the roll tube. In the case of the PYG broth, it may be that a 24-to 48-h culture is too old and that enzyme activity has already begun to diminish due to the presence of acids and possibly toxic end products in the culture medium. Also, the color of the broth may disguise or otherwise compromise the color of the end product in the biochemical reaction. For example, inoculum from PYG broth could not be used to determine the ONPG reaction, since the deep color of the broth obscured the yellow color produced in a positive reaction. In summary, rapid biochemical testing may be a useful adjunct to the identification of anaerobic bacteria. The fact that these tests can be run under aerobic conditions within 4 h and without the use of elaborate equipment suggests their desirability over the present systems for biochemical testing.

v3-fos

2019-08-18T02:44:26.616Z

2014-01-01T00:00:00.000Z

251214956

s2

New and Interesting Records of South African Fungi, Part VIII Descriptions of the following eight fungi isolated from various habitats are given: Ac'taztomium strumarium Rai, Tewari & Mukerji, from a leaf of Protea sp.; Phoma glomerata (Corda) Wollenw. & Hochapf. from wheat debris; Phoma glu.narum Ell. & Tracy and Phoma capitulum Pawar, Mathur & Thirumalachar from leaf litter of Acacia karroo Hayne; Phoma jolyana Pirozynski & Morgan-Jones from contaminated agar plate; Veronaea botryosa Cifferi & Montemartini, Scytali- dium lignicolum Pesante and Rhinocladiella msnsonii (Castell.) Schol-Schwarz from deteriorated canvas; Cerebella andropogonis Ces. from Lolium multiftorum Lam. spikelets. The identity of this specimen was verified by Dr H. A. van der Aa, Baarn. Netherlands. The characters of this isolate agree closely with the description given by Boerema et al. (Persoonia 4: 53-54, 1965) and Morgan-Jones (C.M.I. Descriptions of Pathogenic Fungi and Bacteria. No. 134). Boerema (loc. cit.) pointed out that this species is strongly influenced by cultural conditions and displays wide variation in its main features, but that it is easily recognised by the characteristic chlamydospores produced in chains and resembling the conidia of Alternaria. It should be noted that typical chlamydospores are not readily observed in very young cultures and that these should not be used for identification purposes (Leudemann. Mycologia 51. 772 -780. 1959 Colonies on potato-dextrose agar slow growing and reaching a diameter of 3,5 cm in 10 days, eventually compact and spongy; aerial mycelium cottony, pure white; reverse colourless or faintly rosaceous. Hyphae hyaline, branched, up to 3,0 /m diam. Pycnidia tardily produced in small numbers, superficial or immersed in the medium, subglobose, at first light brown becoming brown to nearly black, neck incon spicuous or absent, mostly with a single ostiole, variable in size and up to 250 /xm diam. Pycnidiospores hyaline, mainly ovoid, smooth, discharged in greyishwhite opaque mass, 2 ,5-7 ,O x2,0-4,0 /xm. Specimen examined: P.U. Culture Collection, No. 71, isolated from leaf litter of Acacia karroo, Potchef stroom, Transvaal, Jan. 1964. PREM 44862 dried culture on 1,5% malt extract agar. The identity of this specimen was confirmed by Dr G. H. Boerema, Wageningen, Netherlands. The identity of this specimen was verified by Dr G. H. Boerema, Wageningen, Netherlands. According to Boerema et al. (loc. cit.) this species is easily recognised by the single, more or less clavate dictyochlamydospores developing terminally or late rally on the hyphae. It is further pointed out that the size and pigmentation of the pycnidia, pycnidiospores and chlamydospores are markedly affected by the age of the cultures and by the C/N ratio of the medium. These variations have caused considerable confusion in the taxonomy of this group of fungi and should therefore be taken into account in the identification of species. This is the first record of the occurrence of this species in South Africa. -M.C.P. The identity of this specimen was confirmed by Dr G. H. Boerema, Wageningen, Netherlands. According to Boerema (Persoonia 5: 203, 1968) the characters of this species in vitro show wide variation but the fungus is, nevertheless, easily recog nized by the dark, beaked pycnidia, the intercalary dictyochlamydospores and the production of a dis tinct reddish (orange to red-purple) pigment. The C/N ratio of the medium affects the production of pycnidia and chlamydospores-a high ratio favouring chlamydospore production and a low ratio the forma tion of pycnidia (Boerema. Persoonia 5: 203, 1968 Colonies on malt agar slow growing, reaching a diameter of 27 mm in 12 days, velvety-granular, dark grey-olivaceous, reverse similar, no pigment diffusing into the agar. Hyphae smooth, septate, branched, with branches often at right angles and with a septum in the main axis close to the point of branching, slightly wavy with cell walls often irregular and slightly bulging, rarely straight, 2-3 /xm diam., olivaceous-grey. Conidiophores macronematous, mononematous, smooth-walled, septate, slightly wavy, olivaceous-grey, branched or unbranched, arising at right angles on trailing hyphae. often basally septate and constricted at this septum, variable in length. 55,0-300,0x2,0-3,0 /xm. Conidiogenous cells inte grated, polyblastic, restrictedly sympodial, rarely geniculate, scars small and flat, mostly determinate but rarely resumes normal growth resulting in an intercalary conidiogenous area. Conidia solitary, dry. acropleurogenous, oblong-ellipsoid to ellipsoid, rounded apically, rarely fusiform, slightly tapering basally, mostly with a definite hilum, 0-1-septate often constricted at the septa, smooth, thin-walled, olivaceous-buff, 5,0-12,0x2,0-5,0 /xm. coprophila. the only species of Sympodina. to be identical with V. botryosa. The latter as described by Cifferi & Montemartini (loc. cit.) differs from the isolate described here as well as from S. coprophila. in having club-shaped conidiophores with small sterigmata and conidia with pointed ends and no constrictions at the septa. Von Arx (personal com munication) found the present isolate to differ only slightly from the type of V. botryosa. It appears that Subramanian & Lodha's (loc. cit.) diagnosis for S. coprophila is actually more acceptable for V. botryosa than the original description by Cifferi & Montemartini (loc. cit.). Although the conidia of V. botryosa are similar to those of V. simplex, the latter species is distinguished by its less elaborate, shorter, geniculate conidio phores with prominent protruding scars. This is the first record of the occurrence of this species in South Africa. -W.J.J. This is the first record of the occurrence of this genus in South Africa. -W.J.J. This isolate differs in some aspects from the descrip tion given by Schol-Schwarz (loc. cit.). The colony is not slimy, neither were the various spore types, viz Phialophora annellate-type and Cladosporium-type observed. The conidia are also larger than the 2,0-3,0 (5,5)X 1,0-1,5 (2,0) /xm reported by Schol-Schwarz (loc. cit.). However, other characteristics such as proliferation of secondary conidia and the anastomos ing of older conidia agree well with those of R. mansonii. In an extensive study of the genus Cerebella. Langdon (loc. cit.) maintained it as a separate genus with C. andropogonis as the only species and listed 32 synonyms. Schol-Schwarz (loc. cit.), however, placed C. andropogonis in the genus Epicoccum as E. andropogonis because of similarties in conidial ontogeny. Ellis (loc. cit.) did not accept this and maintained C. andropogonis on account of its distinc tive conidial morphology and the production of the cerebriform stroma in vivo. The isolate described here has conidial dimensions which agree with the lower limits given by Langdon (loc. cit.). Considering the variability of this genus as indicated by Langdon (loc. cit.), there should be no doubt about the identity of the isolate. It is readily distinguished from E. purpurascens (fig. 11) by the marked constrictions at the septa, the relatively smooth conidial surface and the smaller and lightercoloured conidia. This species has been recorded in South Africa before. According to the synonyms given by Langdon

v3-fos

2018-04-03T03:34:46.803Z

1974-05-01T00:00:00.000Z

6788133

s2

Campylobacter fetus Subspecies jejuni (Vibrio fetus) from Commercially Processed Poultry Three isolates of the human and animal pathogen Campylobacter fetus ss. jejuni (Vibrio fetus) were obtained from 165 poultry meat samples purchased from local retail stores. Campylobacter fetus (Vibrio fetus) is a causa-tive agent of bovine and ovine abortion, scours in calves, of human It Campylobacter fetus (Vibrio fetus) is a causative agent of bovine and ovine abortion, avian hepatitis, scours in pigs and calves, as well as a cause of human infection. It has been isolated from the intestinal contents and organs of several meat animals, including cattle and sheep (1, 8), pigs (6), chickens, and turkeys (10). Dekeyser et al. (2) recovered the organism from mixed microflora (stools) in humans with diarrhea symptoms. Many investigators feel that the incidence of these infections in man is highly underrated. The mode of transmission of human C. fetus infection is obscure, particularly when the disease is found in individuals living in an urban environment without a history of animal contacts. Campylobacter fetus gastroenteritis in man has been traced to the milk supply (4), consumption of raw beef serum (3), and raw beef liver (11). The present study shows further evidence to indicate a possible food-borne epidemiology for human infection with C. fetus. Campylobacter fetus ss. jejuni was isolated from chicken meat obtained from retail stores (Table 1). This organism has not previously been reported from commercially processed chicken parts. The method of isolation and the selective media containing antibiotics were similar to those used by Shepler et al. (7) for the isolation of C. fetus from bull preputial fluid, and those used by Plastridge et al. (5) for the isolation from bull semen and Smibert (8,10) for the isolation from animal feces. The chicken parts were surface rinsed with nutrient broth (BBL, Cockeysville, Md.) by shaking in polyethylene bags. The broth was then filtered (0.65 ,um) and spread on the surface of a selective medium of Brucella agar (Pfizer, Inc., N.Y.) containing 2 units of bacitracin/ml, 2 gg of novobiocin/ml, and 1 unit of polymyxin/ml. These plates were incubated at 37 C for 3 to 5 days in a microaerophilic atmosphere (5% 0,, 'Present address: Route 1, Box 79, Pembroke, Va. 10% CO2, 85% N2). Suspect colonies were picked on the basis of being unpigmented and having a relatively small diameter (less than 2 mm). These were screened for nonfermentation of glucose, oxidase and catalase production, and morphological appearance under phase microscopy. Preparation of these media is described by Smibert (9). Further biochemical tests were employed to confirm the identity of these isolates (see Campylobacter in Bergey's Manual of Determinative Bacteriology, 8th ed., in press). Contaminants were a significant problem in the isolation and recovery of C. fetus from mixed poultry microflora. Alcaligenes faecalis was particularly annoying. This organism can be easily mistaken for C. fetus after the initial screening procedures since it has similar biochemical characteristics and morphological appearance. The difficulties encountered in the isolation of C. fetus and the selective method of recovery suggest that the incidence of this bacterium as found on retail poultry meat is a minimum value. The isolates were biochemically and morphologically indistinguishable from 12 other C. fetus ss. jejuni strains previously isolated from human and avian disease. C. fetus organisms were shown to be capable of surviving on the surface of chicken meat at refrigeration (3 C) and freezing ( -23.5 C) temperatures for periods long enough to allow the meat to be marketed at the retail level (5 days at 3 C and 20 days at -23.5 C). Since the microorganism has been isolated from other meat animals (1, 6, 8), similar microbiological analyses of meat products such as pork, beef, lamb, or turkey may reveal C. fetus contamination. Appreciation is extended to J. Dekeyser, R. E. Weaver, and R. M. Smibert for supplying the C. fetus strains used in this study. This work was supported in part by funds from the Virginia Agricultufal Experiment Station.

v3-fos

2020-12-10T09:04:12.908Z

1974-06-01T00:00:00.000Z

237233130

s2

Volatile Flavor Compounds Produced by Molds of Aspergillus, Penicillium, and Fungi imperfecti Strains of molds Aspergillus niger, A. ochraceus, A. oryzae, A. parasiticus, Penicillium chrysogenum, P. citrinum, P. funiculosum, P. raistrickii, P. viridicatum, Alternaria, Cephalosporium, and Fusarium sp. were grown on sterile coarse wheat meal at 26 to 28 C for 120 h. The volatiles from mature cultures were distilled at low temperature under reduced pressure. The distillates from traps -40 and -78 C were extracted with methylene chloride and subsequently concentrated. All the concentrates thus obtained were analyzed by gas-liquid chromatography, mass spectrometry, chemical reactions of functional groups, and olfactory evaluation. Six components detected in the culture distillates were identified positively: 3-methylbutanol, 3-octanone, 3-octanol, 1-octen-3-ol, 1-octanol, and 2-octen-1-ol. They represented 67 to 97% of all the volatiles occurring in the concentrated distillate. The following 14 components were identified tentatively: octane, isobutyl alcohol, butyl alcohol, butyl acetate, amyl acetate, octyl acetate, pyridine, hexanol, nonanone, dimethylpyrazine, tetramethylpyrazine, benzaldehyde, propylbenzene, and phenethyl alcohol. Among the volatiles produced by molds, 1-octen-3-ol yielding a characteristic fungal odor was found predominant. In our previous paper (9) a description was presented of different volatiles produced by Aspergillus flavus. The purpose of this study was the identification of the volatiles produced by molds of the group Aspergillus, Penicillium, and Fungi imperfecti. MATERIALS AND METHODS Microorganisms. All the microorganisms studied were isolated from wheat grain and were maintained on Czapek-Dox agar slants at 3 C until used. Culture media and growth conditions. The culture medium used was coarse wheat meal sterilized at 1 atm for 45 min. The wheat meal was moistened to 60% water content and inoculated with conidia of pure culture suspended in physiological saline as described previously (9). After 3 to 4 days the culture medium (1 kg) was collected for the isolation step. Isolation of volatiles for the medium. The isolation of volatiles from the culture medium was carried out by vacuum distillation in an all-glass apparatus (10). The distillation step took 4 h and was done under nitrogen at 5 mm Hg. The temperature of the water bath was 35 C, whereas that of the cold traps in which the distillate was collected ranged from -10 to -80 C. The distillate collected in traps cooled to -40 and -80 C was extracted with CH2Cl2 and concentrated to a volume of 100 uliters (10). Gas chromatography. The separation of the volatile substances in the concentrated distillates was carried out with a Willy Giede model GCHF 18.3 gas chromatograph equipped with a flame ionization detector. The columns were stainless steel (3 m long; inner diameter 3 mm) packed with 15% Carbowax 20 M terminated with terephtalic acidon80to 100-meshacid-washed, dimethyldichlorosilane (DMCS)-treated Chromosorb W. Nitrogen was used as the carrier gas at a flow rate of 20 ml/min. Samples (2 gliters) were applied to the column which was held isothermally at 120 C. The percentage of individual volatiles was calculated from the peak area, the area of all the peaks on the chromatogram serving as the 100% value. Identification of the predominlant volatiles. The volatile compounds occurring in the extracts were separated on a gas-liquid chromatography column and then were identified by coincidence of relative retention times with those of known compounds, by mass spectrometry, chemical modification of the sample, and by olfactory evaluation. For identification purposes a combined apparatus gas chromatograph-mass spectrometer LKB 9000 was used. The columns were glass (3 m long, inner diameter 2.5 mm) packed with 10% Carbowax 20 M terminated with terephtalic acid on 80-to 100-mesh, acid-washed, DMCS-treated Chromosorb W. Helium, at a flow rate 10 to 20 cm3/min, served as the carrier gas. The columns were temperature programmed as follows: 5 C/min in the range 70 to 170 C. The ionization chamber was operated at 10' to 10' torr. A 70-eV source provided the mass spectra. Spectra were recorded from mass 30 to 250 in 100 s. Components were identified by comparing the mass spectra of the unknown to authentic compounds. The functional groups were identified also in the head space above the culture medium. The method of Hoff and Feit modified in this laboratory was used (11). The method is based on the reactions of functional groups of the volatile compounds with chemical reagents. The sensory evaluation was performed by a panel of 2 to 3 members by smelling the effluent from the column. RESULTS AND DISCUSSION The volatiles identified in the concentrated distillates from various strain cultures are listed in Table 1. 1-Octen-3-ol was found to be the main volatile component produced by all the molds studied. Its contents varied from 36.6 to 93.1% of the total volatiles. In this respect the most efficient appeared to be such molds as P. citrinum, P. raistrickii, Cephalosporium, P. funiculosum, and A. niger. Pure 1-octen-3-ol isolated from the molds by a gas chromatography trapping procedure exhibited a strong fungal resinous odor. In concentrations close to the threshold value its odor resembled that of the mushroom, Agaricus bisporus. According to the data presented in Table 1, the quantity of 1-octen-3-ol depends on the mold species and composition of the growth medium. Thus, A. ochraceus grown on such media as coarse wheat meal, starch, gluten, and plant oil was stated to produce volatile fractions differing both qualitatively and quantitatively. The contents of 1-octen-3-ol were calculated by measuring the peak area on chromatograms of the concentrated distillates. This strain was found to produce the highest amounts of 1-octen-3-ol when grown on coarse wheat meal (83.8%); lower yields were obtained on gluten (26.9%), starch (9.3%), and only traces on media containing plant oils. It should be stressed that the uninoculated coarse wheat meal contained only traces of the above mentioned compounds (9). A research was also carried out in this laboratory on the mushrooms: Agaricus bisporus and Boletus edulis (14,15). According to the data obtained, 1-octen-3-ol was the predominant volatile in Agaricus bisporus and B. edulis-78 and 82.5% of the total volatiles, respectively. It is noteworthy that this compound occurs in many other foods; its origin has not been established, however (1, 2, 4,5,7,8,12,13). According to the results of mass spectrometry and infrared analysis, 1-octen-3-ol produced by molds and mushrooms is identical (14,15). Thus, a number of strains can be used to produce foods showing the odor typical of mushrooms. In Table 2, data are presented on the contents of 1octen-3-ol in the head space above Agaricus bi- sporus and above the coarse wheat meal on which molds were cultivated. For A. oryzae, the vapors above the substrate contained 200 to 300 times lower quantities of 1-octen-3-ol as compared with those above the freshly harvested mushrooms, but more than in boiled ones. In the case of Cephalosporium, the level of 1-octen-3-ol in vapors was found to be only 2 to 3 times lower than in vapors above the fresh mushrooms. Certain mold species, for example A. oryze, are used in the production of soybean products (3,6). The mold species studied were found to be capable of producing other volatiles also (Table 1). Among the latter compounds, of special interest is 2-octen-1-ol, which yields a characteristic, unpleasant musty-oil odor. Some mold species, for example A. flavus (9) and A. parasiticus, produce this compound in large quanities. There are other mold strains, however, which produce 2-octen-1-ol in small amounts. Besides the compounds presented in Table 1, the following volatiles were identified tentatively by chemical reactions and mass spectrometry: octane, isobutyl alcohol, butyl alcohol, butyl acetate, octyl acetate, pyridine, hexanol, nonanone, dimethylpyrazine, benzaldehyde, propylbenzene, and phenethyl alcohol. Although the above compounds occur in low concentrations, they affect considerably the flavor of foods infected by molds. The volatiles produced by molds may be used as an index for the detection of food contamination by rapid instrumental methods.

v3-fos

2020-12-10T09:04:12.839Z

1974-01-01T00:00:00.000Z

237234520

s2

Evaluation and Modifications of Media for Enumeration of Clostridium perfringens The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfitecycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. SPS agar selectively inhibits growth or interferes with the formation of black colonies by the sulfite-reducing Enterobacteriaceae and Achromobacteriaceae; it also inhibits growth of most other facultative anaerobes and of the genera Pseudomonas, Bacillus, and Lactobacillus (1,5). However, low recoveries of C. perfringens in commercial SPS agar have been reported (12; L. F. Harris and J. V. Lawrence, Bacteriol. Proc. 70: 6,1970). Hauschild et al. (7) recovered 12 strains of C. perfringens quantitatively in SPS agar prepared in the laboratory from its ingredients, but in only one out of four commerical lots of SPS. Handford and Cavett (4) and Harmon et al. (5) also obtained low recoveries in laboratory-prepared SPS agar. In this laboratory, we have usually obtained complete recoveries of C. perfringens in SPS agar prepared from its ingredients, but in two preparations the recoveries of some C. perfringens strains were below 1% (D. Dobosch and A. H. W. Hauschild, unpublished data). In one preparation, the cause could be traced to a particular lot of yeast extract. It appears that the selective ingredients of this agar are at a level where a slight adverse change in the medium may result in inhibition of C. perfringens. TSN agar has been used less extensively than SPS agar, but the few reports on the suitability of this medium indicate that it is inhibitory to a number of C. perfringens strains (4, 5; Harris and Lawrence, Bacteriol. Proc. 70: 6,1970). SFP agar appears to allow quantitative recovery of C. perfringens (4)(5)(6)12). Unfortunately, it does not prevent growth of a large number of facultative anaerobes, some of which are sulfite reducing (6,12). Its applicability, therefore, seems to be limited to specimens in which C. perfringens is the predominant microorganism, i.e., foods responsible for C. perfringens enteritis or fecal samples from patients recovering from the disease. The use of neomycin-blood agar commonly used in the United Kingdom (8,14) is similarly limited to investigations of food-poisoning incidents. Another disadvantage of the SFP agar is its relatively elaborate preparation: it requires addition of fresh egg yolk, surface plating, and pouring of cover agar. Harmon et al. (6) modified SFP agar by replacing polymyxin B and kanamycin with 0.04% D-cycloserine. This antibiotic had been shown to selectively inhibit growth of essentially all of the common facultative anaerobes (2). In this modified medium (TSC agar), each of 10 C. perfringens strains tested was enumerated quantitatively by Harmon et al. (6). Presumptive enumeration of C. perfringens is followed by confirmatory tests. The simplest of these involves stab culturing of an adequate number of black colonies into nitrate motility (NM) agar (1). However, the nitrite test as described by Angelotti et al. (1) is unreliable (3,12). Shahidi and Ferguson (12), therefore, intro-78 duced egg yolk into their medium and proposed to enumerate only black colonies with an opaque halo around them and to confirm these in lactose motility (LM) agar. Of the clostridial species that produce sulfide as well as lecithinase, only C. perfringens is nonmotile and lactose positive. ln our experience, this method has the following main shortcomings: (i) several C. perfringens species do not produce a discernible halo after 20 to 24 h of growth in SFP and TSC agars; (ii) due to excess gas formation in LM agar, nonmotility of these isolates is difficult to ascertain. This work was initiated to evaluate the suitability of the SFP and TSC agars for enumeration of C. perfringens and to determine the conditions required to obtain consistent results in the nitrite motility test. While this work was in progress, Handford and Cavett (4) published a note on the enumeration of C. perfringens in OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agar. An evaluation of this medium is included in the present paper. MATERIALS AND METHODS Cultures. Seventy-one strains of C. perfringens were examined; 51 of these were isolated from foodpoisoning incidents, 11 from pathological specimens, 7 from soil and normal feces, and 2 were of unknown origin. Strains were supplied by C. R. Amies, Willowdale, Ontario (six strains); R. J. Avery, Hull, Quebec The working cultures were preserved in 15% glycerol (10) at -18 C; they were thawed, inoculated into screw-cap test tubes containing 15 ml of cooked meat medium (Difco), and incubated at 37 C for 20 h. Enumeration procedures. The cultures were diluted in 0.1% peptone (13). When egg yolk-containing media were used, 0.1-ml volumes of diluted culture were spread on the agar surface in standard petri plates. Two plates were used per dilution. When completely dry, the surface was covered with about 10 ml of cover agar. Egg yolk-free media were used in pour plates with 1.0-ml volumes of diluted culture per plate. All plates were incubated anaerobically at 37 C for 20 h. All plating media were prepared from the same agar base consisting of 1.5% tryptose (Difco), 0.5% Soytone (Difco), 0.5% yeast extract, 0.1% ferric ammonium citrate (British Drug Houses), 0.1% sodium metabisulfite (Na2S2O5; British Drug Houses), and 2% agar. The ingredients were dissolved in distilled water to either 92% of the final volume to allow for subsequent addition of egg yolk suspension, or to the final volume. The pH was adjusted to 7.6 before addition of the agar. The agar base was also obtained commercially (SFP agar base, Difco). Antibiotics and egg yolk suspension were added to the autoclaved medium at 50 C. SFP agar. Complete SFP agar was prepared by adding to 920 ml of agar base: the contents of one antimicrobial vial P (30,000 U of polymyxin B [Difco] in 10 ml of distilled water); 4.8 ml of the contents of an antimicrobial vial K (25 mg kanamycin [Difco] in 10 ml of distilled water); and 80 ml of egg yolk suspension containing one egg yolk per 20 ml of 0.85% NaCl. The SFP cover agar had the same composition as the complete SFP agar, except that it contained no egg yolk. Media with D-cycloserine. The second group of plating agars contained varying amounts of D-cycloserine (D-CS; Nutritional Biochemical Corp., Cleveland, Ohio) instead of polymyxin B and kanamycin (6). The medium containing 400 gg of D-CS per ml (0.04%) is identical with the TSC agar of Harmon et al. (6). The antibiotic was added as a 4% filter-sterilized solution in water. The plating procedure was as described for the SFP agar. The third group of plating agars differed from the second in two aspects: no egg yolk was added, and they were used in pour plates only. OPSP agar. Details for the preparation of OPSP agar not contained in the note of Handford and Cavett (4) were obtained by personal communication. The basic ingredients, including ferric ammonium citrate and sodium metabisulfite, were the same as in the SFP and TSC agars. The final concentration of sodium sulfadiazine 272 molecular weight (American Cyanamid Co., Pearl River, N.Y.) was 109 mg/liter, which corresponds to 0.01% sulfadiazine (250 molecular weight) used by Handford and Cavett. Concentrations and origins of the antibiotics were the same as in the work of these authors; the final concentrations of oleandomycin phosphate (Pfizer Co., Montreal) and polymyxin phosphate (aerosporin; Burroughs Wellcome Co., Montreal) were 0.5 mg/liter and 10,000 IU (equivalent to 1.0 mg of polymyxin standard) per liter, respectively. The OPSP agar was used without egg yolk and in pour plates. The results of all enumerations were expressed as percentages of the counts in the corresponding antibiotic-free control medium. Confirmatory tests. Single colonies were stabinoculated into LM agar (12), NM agar (1), and NM agar supplemented with 0.5% each of glycerol and galactose (11) (12) and of Harmon et al. (5,6), but the experiment revealed two considerable shortcomings of both media. (i) Most C. perfringens strains produced large colonies in SFP and TSC agars; counts of over 50 per plate therefore became progressively inaccurate, and 10-fold dilutions were often inadequate. (ii) Of 21 strains, 8 had no discernible opaque halos around the black colonies after the first day of incubation; presumably, such colonies would not be counted in the procedure of Shahidi and Ferguson (12). These drawbacks as well as the lengthy plating procedure are all associated with the dependence of the method on the egg yolk reaction which had been introduced because the nitrite motility test was unreliable. The following experiments were designed to determine the conditions (i) for quantitative enumeration of C. perfringens in a selective medium without egg yolk and (ii) for consistent nitrite reactions in the confirmatory tests. Enumeration in egg yolk-free agar with D-CS. Table 1 shows the recoveries of 71 strains in egg yolk-free agar with different concentrations of D-CS. The recoveries were essentially quantitative at D-CS concentrations of 200 and 400 jig/ml; the lowest count at 400 Ag/ml was 64% of the count in the control medium. Several strains were partially or totally inhibited at D-CS concentrations of 600 and 800 jig/ml, with mean recoveries of 63 and 39%, respectively. About 40% of the strains listed in Table 1 were tested in medium with the commercial agar base; due to supply problems, the remaining strains were tested in medium prepared from the ingredients. This did not appear to affect the results. A detailed table showing the recoveries of each strain is available upon request. At all concentrations of D-CS (Table 1), the strains produced only black colonies, even at the surface. However, a few strains occasionally produced colonies at the surface with a narrow, white halo around the black center. The medium with the highest D-CS concentration (400 jig/ml) that does not significantly Table 2. Of the 142 isolates (duplicate colonies of the 71 strains), only 112 showed a positive nitrite reaction in basic NM medium. Five tubes contained traces of nitrite as evidenced by a faint red color, and 25 tubes contained no detectable nitrite. Most of the negative tubes showed only little growth. All of the isolates grown in supplemented NM medium showed good growth and produced positive nitrite reactions; most of these were very intense, in contrast to the reactions in basic NM medium ( Table 2). Two strains each of C. sporogenes and C. bifermentans were used as negative controls; none of these showed a color reaction. During incubation, the pH of the C. perfringens stab cultures dropped from 7.1 to 6.7-6.9 in NM agar and to 5.6-6.1 in supplemented NM agar. Uninoculated tubes with supplemented NM agar, adjusted to pH 5.5, were therefore incubated as additional controls; they were all negative for nitrite. All NM media were used within 2 weeks after preparation, and all were de-aerated before stabbing. In a separate experiment, we compared the suitability of freshly prepared, supplemented NM medium with the same medium stored for 5 weeks at 4 C. No difference was found. In contrast, basic NM medium deteriorated rapidly during storage: in the fresh medium, 24 out of 28 isolates showed positive nitrite reactions, and 4 gave trace reactions; in the same medium stored for 3 weeks at 4 C, only two isolates produced nitrite; the remaining 26 were negative for nitrite. Comparison of surface-plated egg yolk media with egg yolk-free pour media. We compared the recoveries of 19 C. perfringens strains in the presence of D-CS in pour plates without egg yolk (as in Table 1) and in surfaceplated medium with egg yolk and cover agar (6). The SFP medium was included for comparison. In both media containing 400 Ag of D-CS per ml and in SFP agar, the recoveries were essentially quantitative (Table 3). At the higher concentrations of n-CS, the recoveries in medium with egg yolk were considerably lower than in egg yolkfree medium. The differences were likely due to exposure of the C. perfringens cells to high oxygen tension in the surface plating procedure. The results demonstrate that the recoveries of C. perfringens in the proposed procedure are equal to or higher than recoveries in the existing procedures. In this work, we have not tested the selective inhibition of single strains of facultative anaerobes by the EY-free TSC agar. However, applications of this medium for enumeration of C. perfringens in naturally contaminated foods and in fecal specimens (A. H. W. Hauschild and R. Hilsheimer, manuscript in preparation) have shown essentially the same degree of selectivity as that of the egg yolk medium of Harmon et al. (6). Some shortcomings of the media containing egg yolk have been listed above: (i) the low selectivity of SFP agar; (ii) the relatively elaborate procedures; (iii) the frequent occurrence of C. perfringens colonies without discernible halos (false negatives); and (iv) the large and frequently spreading colonies which make 10fold dilutions impractical. We have also found that SFP agar allows growth of egg yolk-positive facultative anaerobes from foods. In some cases, these organisms produced completely opaque plates and thus masked the egg yolk reaction of C. perfringens. The lack of selectivity of the SFP agar has been overcome by replacing it with TSC agar (6). The remaining shortcomings of the egg yolk agars may be overcome by using EY-free TSC agar in pour plates and stab-culturing black colonies of supplemented NM agar for confirmation of C. perfringens. Comparison of D-CS from different sources. Since we did all of our work with D-CS from a single supplier (Nutritional Biochemicals Corp.), its effect on the enumeration of C. perfringens was compared with that of 1-CS from another company (Sigma Chemical Co., St. Louis, Mo.). Five C. perfringens strains were tested. Essentially the same results were obtained with -CS from both suppliers ( Table 4). Comparison of EY-free TSC agar with OPSP agar. Table 5 shows the recoveries of 22 C. perfringens strains in EY-free TSC and OPSP agars. As in preceding experiments (Tables 1 and 3), the recoveries of all strains were essentially quantitative in EY-free TSC agar. Twenty of these strains were also enumerated quantitatively in OPSP agar, but one of them (8247) produced only pin-point colonies that c Only pin-point colonies were produced. were difficult to count even in pure culture. A few of the other 19 strains, namely 1194 and 11668, had relatively small colonies, but these could still be counted without difficulty. For the remaining two strains (A-72, S-88), the counts were low; no colonies of strain A-72 were found, and strain S-88 had pin-point colonies only. Handford and Cavett (4) obtained quantitative recoveries of C. perfringens in OPSP agar, but they tested only eight strains. Our work with naturally contaminated foods (A. H. W. Hauschild and R. Hilsheimer, manuscript in preparation) has shown that facultative anaerobes were considerably less inhibited in OPSP agar than in the TSC agars. The OPSP agar, therefore, is not satisfactory for enumeration of C. (9) by replacing its antibiotics with D-CS. The modified medium has not as yet been thoroughly tested.

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2018-04-03T01:22:50.170Z

1974-01-01T00:00:00.000Z

29140057

s2

Antibody to viruses affecting cattle in commercial tissue culture grade fetal calf serum. Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum. Received.for publication 24 September 1973 Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum. Under normal conditions immunoglobulins are transferred from a cow to its calf solely via colostrum during the 1st day of a calf's life. There appears to be no evidence for intrauterine transfer as occurs in many other species (4). As the bovine fetus matures, it gradually develops immunocompetence so that it is able to respond to certain antigenic stimuli beginning at about the 118th day of gestation (16). Bovine viral diarrhea (BVD) (1, 10), bluetongue (3,12), and infectious bovine rhinotracheitis (IBR) (5,9) viruses have been reported to infect the bovine fetus by traversing the placental barrier. Antibody to these viruses has been found in infected fetuses (10,13,15). Recently, Hubbert et al. (8) tested the sera of apparently normal fetuses for antibody to the bovine strain of parainfluenza 3 (PI3-SF4), BVD, and IBR viruses. Antibody to these viruses is found in the majority of adult cattle; however, they only found antibody to BVD virus in 3 of 147 fetal sera and no antibody to either PI3-SF4 or IBR virus in about 100 fetal sera. Horner et al. (7) also tested the sera of individual fetuses. By using a variety of infectious agents as antigens, they found antibody only to BVD virus in a small percentage of the sera. Because of its availability, its growth promoting capacity, and its absence of immunoglobu-IPublication no. 1160, School of Veterinary Medicine, Auburn University, Auburn, Ala. 36830. lins, fetal calf serum (FCS) has been widely used in culturing cells of many species. However, Kniazeff et al. (11), noting that many investigators found that pooled FCS neutralized certain viruses, tested individual sera from bovine fetuses in their 4th to 6th month of gestation. They found that a few fetuses had antibody to BVD, but not to IBR or PI3-SF4 virus. The antibody detected in the fetus was attributed to either fetal synthesis or to transplacental transfer. Considering present information, transplacental transfer seems to be a remote possibility for explaining the presence of antibodies in the serum of bovine fetuses. Investigations of Boone et al. (2) showed that different lots of commercial FCS for use in tissue culture varied considerably in chemical composition. They suggested that high gamma globulin levels may be associated with adulteration with postcolostrol serum. In our work with bovine viruses, we have used the micro-serum neutralization (micro-SN) test in which virus and test serum are mixed and incubated together. Cells are seeded in this mixture so that this procedure requires a concentration of about 10% serum for suitable cell growth. Because of the requirement of serum for cell growth, antibody in the culture medium is a significant factor as compared with neutralization tests conducted on confluent cultures in which the concentration of serum in the medium is minimal. This report concerns the examination of commercial FCS for antiviral inhibitors to viruses affecting cattle to determine the suitability of different types and lots of FCS for use in the micro-SN system. MATERIALS AND METHODS Test serum. All tissue culture sera tested were from different serially numbered lots purchased from Grand Island Biological Company, Grand Island, N.Y., during the interval from the autumn of 1970 to the spring of 1973. The sera were heat inactivated nrid stored at -20 C until tested. The sera were identified by the manufacturer as (i) FCS, (ii) virus-screened FCS (VS-FCS), (iii) immunoprecipitin-tested FCS (IPT-FCS), or (iv) gamma globulin-free newborn calf serum (GG-free NCS). The latter two sera were subjected to a modified Cohn fractionation (6) procedure by the manufacturer to remove gamma globulins. Viruses. The following viruses were used in the micro-SN test: IBR, PI3-SF4, bovine adenovirus type 1, bluetongue strains 8 and OX 183 (BT 8 and BT OX 183), the New Jersey serotype of vesicular stomatitis (VS), and two bovine enterovirus strains identified as ED-1244 and 66-P-188 (supplied through the courtesy of J. Storz, Colorado State University, Fort Collins, Colo.). Micro-SN test. The micro-SN test was conducted as previously described (14) by using 50 to 100 mean tissue culture infective dose of virus, except for the following modifications. Medium for cells in microtiter consisted of Eagle minimal essential medium with modified Earle salts (Grand Island Biological Co.) and 10% IPT-FCS. The medium was buffered with 0.2% NaHCO3, 7.5 mM N-2-hydroxyethylpiperazine-N'-2'-ethane-sulfonic acid, and 5 mM each of TES and MOPS. Each serum was tested in triplicate by using serial twofold dilutions and starting with undiluted serum. A serum toxicity control was included for each dilution of serum. Complement enhancement test. Several sera which showed some inhibition of IBR virus, a herpesvirus, were tested with and without complement diluted 1:3 as described by Yoshino and Taniguchi (17), except that serum-virus-complement mixtures were assayed in microtiter by using six replicates per dilution. Complement has been found to enhance detection of antibody to IBR virus in certain bovine sera (C. R. Rossi and G. K, Kiesel, unpublished data). Cells consisted of a strain of bovine embryonic lung cells for assay of VS, IBR, BT 8, BT OX 183, and P13-SF4 viruses and the AU-BEK cell line, established in this laboratory (C. R. Rossi and G. K. Kiesel, In Vitro, in press), for assay of bovine adenovirus type 1 and bovine enteroviruses 66-P-188 and ED-1244. RESULTS AND DISCUSSION Results with the micro-SN test for detecting neutralizing substances in commercial sera against viruses which affect cattle are shown in Table 1. Low neutralizing titers against VS virus were found in most of the sera tested. The presence of neutralizing substances in IPT sera indicates the activity was not associated with the gamma globulin fraction of serum and suggests its non-antibody, nonspecific nature. Excluding VS virus, the five IPT-FCS tested did not inhibit replication of any other virus. At least two lots of IPT-FCS were toxic enough to prevent attachment and growth of bovine embryonic lung and AU-BEK cells when used at normal concentrations. This toxicity could be removed to allow satisfactory cell growth in the microtiter system by heating the serum at 56 C for 0.5 h. The GG-free NCS, one lot of FCS, and two lots of VS-FCS had inhibitory activity to several of the viruses, whereas most lots were completely free of any viral inhibitory activity. Explanations which can be offered for the presence of viral inhibitory activity in the commercial sera tested include the presence of (i) nonspecific inhibitors, (ii) fetal antibodies, (iii) natural antibodies, or (iv) antibodies due to contamination with post-colostral bovine serum or, in the case of GG-free NCS, ingestion of colostrum and incomplete removal of immunoglobulins. Serum lots 1, 3, 10, and 12 had inhibitory activity to viruses from several taxonomic groups, whereas most other lots of serum had no neutralizing activity against any virus, except for that against VS virus as previously mentioned. The distribution of neutralizing activity to several lots and types of sera tends to implicate antibody as the neutralizing substance detected against PI3-SF4, adenovirus type 1, and the two enteroviruses. Enhancement of neutralization to IBR virus by complement further indicates the specificity and antibody character of the neutralizing substances. The source of the antibody in these sera is probably contamination with serum from postcolostral bovine serum, in which these antibodies are common. The probability of fetal synthesis or natural antibody being distributed in the sera, as evidenced by the distribution of neutralizing activity in the sera, is remote. Because antibody was found to most of the test viruses in those lots of serum which contained antibody, it is likely that antibodies to many other viruses were also present in these same lots. Therefore, the micro-SN test would appear to be a useful technique for screening lots of commercial serum for antibody to bovine viruses and as an aid in identifying serum contaminated with post-colostrol bovine serum. The use of a few widespread bovine viruses should be appropriate in detecting such contamination. adenoof para in-Vesicular bovine OX virus 66-P-ED-its hiora ACKNOWLEDGMENT This investigation was supported by the Alabama Agricultural Experiment Station, Auburn, Ala.

v3-fos

2020-12-10T09:04:20.903Z

1974-12-01T00:00:00.000Z

237233767

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Membrane Concentration of Infectious Bovine Rhinotracheitis Virus from Water A membrane adsorption procedure was used to concentrate infectious bovine rhinotracheitis virus from 1-liter quantities of distilled water. Cellulose nitrate membrane filters (0.45-μm pore size) efficiently adsorbed this herpesvirus from water, and virus was recovered from the membrane by elution with 10 ml of fetal calf serum during sonic treatment. The average recovery rate was 70%. Infectious bovine rhinotracheitis (IBR) is a disease of cattle that produces upper respiratory inflammation as well as several other syndromes, the most economically important of these being abortion (6). Diagnostic investigations for a 4-month period in 1971 indicated that 67.8% of the 370 fetuses submitted to the North Dakota State Veterinary Diagnostic Laboratory were aborted due to IBR (3). The purpose of this investigation was to develop an IBR virus concentration procedure which could possibly be used to determine if this virus is present in livestock drinking water. The virus concentration methodology developed for this purpose was modified after numerous reported virus concentration techniques (1,4,5,8,9). (This study represents a portion of a thesis submitted by S. R. T. in partial fulfillment of the requirements for the M. S. degree in bacteriology at North Dakota State University, Fargo, 1974.) MATERIALS dispersed with 100 ml of trypsin (0.25%) containing 0.2% ethylenediaminetetraacetate, and the cell suspensions were centrifuged for 10 min at 500 x g to pellet the cells. The cells were then suspended in minimal essential medium containing 10% calf serum and antibiotics. The cells were cultured in disposable tissue culture dishes (60 by 15 mm) at 37 C in a CO2 incubator until confluent monolayers were formed. The minimal essential medium was then removed, and the monolayers were inoculated with 0.5-ml volumes of virus suspensions and the agar was overlaid (minimal essential medium with 1% ion agar). Upon development, plaques were resolved by staining the cells with a 1:1,500 neutral red solution. Membrane concentration procedure. One-milliliter volumes of an IBR virus suspension (4 x 104 PFU/ml) were diluted 1:1,000 in acetate-buffered (0.1 M, pH 6.5) water samples containing MgCI2 (0.5 M). The addition of MgCl2 was necessary to prevent virus loss during filtration (9). The samples were then filtered through type HA membrane filters (47-mm diameter, 0.45-,gm pore size; Millipore Corp., Bedford, Mass.) to adsorb the virus. Virus was released by suspending the filters in a 10-ml volume of fetal calf serum which was then sonically treated in an ice bath with the standard probe of a Biosonic IV sonic oscillator (Bronwill Scientific, Rochester, N.Y.) at maximum output. RESULTS AND DISCUSSION No IBR virus was detected in any of the type HA membrane filtrates, indicating complete virus retention. An increase in adsorption pH from 3.0, a recommended pH for the adsorption of enteroviruses (5), to 6.5 maintained the viability of this herpesvirus yet did not impede virus adsorption. Recovery of the adsorbed viruses was accomplished by sonically treating the membrane filters in 10 ml of fetal calf serum. To determine the amount of sonic treatment required for optimum IBR virus release, membranes were sonically treated for 2-to 5-min intervals. Maximum virus PFU were 1030 obtained after 3 min of sonic treatment ( Table 1). The inactivation of IBR virus was determined by sonically treating virus in fetal calf serum alone; virus was added to 10-ml samples and sonically treated for various times. The recovery rates were combined with the virus inactivation curve (Fig. 1) to indicate that, although 100% of the virus was released after 3 min of sonic treatment, 30% of the virus was inactivated. Although an average virus PFU recovery rate of 70% was obtained, the standard error of the mean was 9.4%. This variation within replicates indicates why there were no significant differences among mean recovery rates listed in Minutes of Sonication FIG. 1. Effects of increasing sonication on IBR virus recovery from type HA membrane filters suspended in 10 ml of fetal calf serum (0) and on the recovery of IBR viruses diluted in 10 ml of fetal calf serum (0). The membrane concentration technique was found more effective in recovering the IBR virus than were three other procedures (S. R. Tschider, M. S. thesis, North Dakota State Univ., Fargo, 1974). Polyelectrolyte 60 (11) and aluminum hydroxide (10) adsorption procedures were found to effectively remove virus from water samples, but the inability to recover IBR virus from these adsorbents restricted their usefulness. The aqueous polymer two-phase procedure that was utilized, a modification of the procedures of Shuval et al. (7), was found to be unacceptable. The two-phase virus concentrates were cytotoxic, and therefore the detection of virus PFU was not possible. All methods examined were modified from the originally reported procedures. Differences in virus characteristics (e.g., pH stability) between the enterovirus group and the herpesvirus necessitated these modifications. In summary, the membrane procedure was found to be an efficient method for obtaining 100-fold concentration of the IBR virus from water, with a recovery efficiency of 70%. Not only is this technique applicable to the concentration of IBR virus, but it also provides a procedure which could be used to concentrate other herpesviruses. Presently, this technique is being investigated for use in evaluating supplies of North Dakota livestock drinking water.

v3-fos

2020-12-10T09:04:12.209Z

1974-04-01T00:00:00.000Z

237231254

s2

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 × 1010 to 7.64 × 1010 per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10-9) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed. Numerous studies on the intestinal microflora of the domestic fowl, Gallus domesticus, have been made (2,3,5,23,26,30,34). In many of these investigations, selective plate media and conventional anaerobic jar methods have been used in an analysis of specific bacterial groups, namely, coliforms, streptococci, lactobacilli, bacteroides, and clostridia. At best only these groups of bacteria have been identified, and unless very good anaerobic techniques were used, some of the predominating anaerobic species have been overlooked. Others, however, have developed nonselective media for the isolation of rumen anaerobic bacteria (9,12). With these media (containing low levels of energy sources) in conjunction with strict anaerobic techniques, numerous bacterial types are permitted to grow. Relatively few studies on poultry intestinal microflora have been made using such media and methods. Recent work by Barnes et al. (3,5) on the isolation of anaerobes from the chicken cecum has indicated that 20 to 678 30% of the direct microscope count can be cultured by using the Hungate technique and a nonselective medium such as medium 10 (medium without rumen fluid devised for rumen anaerobes by Caldwell and Bryant [12]) supplemented with liver and chicken fecal extracts. However, data on comparison of other roll-tube media (e.g., rumen fluid-containing media used for culturing the predominant ruminal species) or the effect of various medium constituents on the enumeration and isolation of chicken cecal bacteria are not available. Experiments in this laboratory indicate that a rumen fluid medium can be used to isolate a large percentage of the total bacteria from chicken cecal digesta. In addition, our present studies compare the cultural characteristics and isolation efficiency of other nonselective rolltube media and furnish information on the presumptive identification and relative distribution of the predominant anaerobic bacteria colonizing the cecum of the chicken. MATERIALS AND METHODS Animals and diet. In experiments designed to determine colony counts, recovery of anaerobes, and effects of medium components in different nonselective media, cecal samples were obtained from 5-weekold cockerels (White Cornish cockerel x White Rock hen) supplied by a commercial broiler facility. In all other experiments on the cecal microflora including isolation and identification of bacterial strains, cecal samples were obtained from 5-week-old, "laboratoryreared" birds described below. One-day-old cockerels (same breed as above) were housed in temperature-controlled rooms (12-h light cycle and slight negative pressure) on floors covered with autoclaved pine shavings. An initial brooding temperature of 35 C was maintained for young birds and reduced in weekly increments to a constant temperature of 25 C at 3 weeks of age. Throughout the growth period, a commercial broiler grower ration and water were provided without restriction. The diet was supplemented with a coccidiostat continuously and with antibiotic (50 mg of bacitracin per kg of feed) from day 1 to 4 weeks of age. After 4 weeks, antibioticfree ration of the same composition replaced the medicated ration. Anaerobic culture techniques. Anaerobic techniques employed were similar to those described by Hungate (20,21) for rumen bacteria with modifications of Bryant (6,7). Strict anaerobic techniques were maintained throughout all procedures involving the dilution of cecal samples and preparation and inoculation of media. Sampling procedure. Five-week-old cockerels were taken at midday and sacrificed by CO2 asphyxiation. Immediately, cecal samples (2 to 4 g, wet weight) from both ceca of a single animal were placed in a sterile, stoppered tube and flushed with oxygen-free CO2. Cecal contents were weighed and processed through serial 10-fold dilutions in tubes of the anaerobic mineral solution of Bryant and Burkey (7). Fractions (0.2 and 1.0 ml) of a dilution (up to 10-9) were added to prereduced and melted agar tubes of the various media maintained at 48 C. Within 10 min after inoculation, roll tubes were made by rapidly rotating tubes in a spinner (Bellco Glass, Inc.) and simultaneously cooling with cold water. Solidified agar roll tubes were incubated up to 14 days at 37 C. Roil-tube media and media preparation. The compositions of different nonselective roll-tube media compared in this study are given in Table 1. Medium 98-5 is an improved rumen fluid medium for culturing ruminal bacteria and has been described by Bryant and Robinson (9). Modified 98-5 (M98-5) medium is one used by R. Williams and M. P. Bryant for culturing bacteria from anaerobic sludge digesters (unpublished data) and is different from 98-5 medium in that glycerol, Trypticase, and hemin are included and mineral solution S2 is substituted for mineral solutions 1 Medium 10 (M10) was developed by Caldwell and Bryant (12) and contains a mixture of volatile fatty acids, hemin, Trypticase, and yeast extract in place of rumen fluid. Supplemented M10 as described by Barnes and Impey (3) contains liver extract (5%, vol/vol) and chicken fecal extract (10%, vol/vol) added to M10. Liver extract (3) was prepared by heating a 13.5% (wt/vol) solution of dehydrated liver (Difco Laboratories) at 50 C for 1 h. The extract was clarified by centrifugation at 10,000 x g, adjusted to neutral pH, sterilized by autoclaving, and stored In addition to these culture media, two types of blood agar media were evaluated with respect to recovery of total anaerobes from cecal material: VL blood (oxalated horse blood) agar as described by Barnes and Impey (3) and Schaedler blood (defibrinated rabbit blood) agar of Starr et al. (32). These media were equilibrated and tubed under CO2 gas phase. Blood agar, prepared in this way, supported hemolysis of known hemolytic bacteria and therefore contained intact red blood cells. In the preparation of all roll-tube media, components except Na2CO, buffer, reducing agent (cysteine or cysteine-sulfide), and blood were diluted to volume in round-bottom flasks, equilibrated with CO2 gas by gentle boiling, fitted with rubber stoppers, and sterilized by autoclaving (15 psi, 15 min). After autoclaving, a sterile reducing agent, buffer, or blood was added to media held at 48 C and then dispensed (9-ml amounts) anaerobically into sterile, rubber-stoppered, disposable tubes (18 by 150 mm, Bellco Glass, Inc.). Direct microscope counts, colony counts, and statistical analysis. Direct microscope cell counts were made on cecal samples employing a Petroff-Hauser bacterial counting chamber and a phase-contrast microscope following the method of Meynell and Meynell (25). For microscope counts, cecal sample dilutions of 10-i were made in 0.9% saline containing 10% Formalin. Colony counts in roll tubes were determined after 3, 6, and 14 days of incubation with the aid of a Quebec colony counter. Colony counts from eight roll tubes (prepared from a single dilution) were determined using the colony-counting criteria of Bryant and Robinson (9). Mean counts and standard errors from several cecal samples were obtained, and the data were subjected to an analysis of variance and differences between means evaluated with the 5% least significant difference (31). In some cases, colony counts were converted to percent recovery of the direct microscope counts. Isolation of cecal bacteria and presumptive identification of strains. Fifty isolated colonies were picked from single roll tubes of M98-5 (containing usually 50 to 100 colonies) inoculated with dilutions of cecal contents and incubated for 6 days. About 300 colonies were isolated from single cecal samples of six different birds. Isolates were subcultured onto maintenance slant medium (composition as modified 98-5, Table 1, except glucose, cellobiose, and maltose were at 0.05% wt/vol concentration). Wet mounts of each isolate, prepared from the water of syneresis of slant media (24to 48-h cultures), were observed for morphology, motility, and purity with phase-contrast microscopy. These same cultures were gram stained according to the Kopeloff modification (19). Isolates containing more than one morphological type were separated, and strains were reisolated from roll streaks (19) of M98-5 medium. Pure cultures of strains were grouped and presumptively identified by using media and methods described by Caldwell and Bryant (12). Oxygen relations (facultatively anaerobic or anaerobic) of isolated strains were determined on aerobic plate media. Cultures (by loopful) were streaked onto brain heart infusion-blood agar described by Holdeman and Moore (19), and surface growth was observed at 1, 3, and 6 days of incubation at 37 C. In addition to these preliminary tests, fermentation products elaborated in glucose medium (11) were analyzed for volatile and nonvolatile acids. Selected strains from each of the presumptively identified groups were then processed through carbohydrate fermentation and biochemical tests. Organisms were identified according to classification schemes of Holdeman and Moore (19) and by comparison with other published data on anaerobic bacteria. The basal medium for carbohydrates and substrates fermented in these studies was that of Bryant (11) and contained the following components: 20% (vol/vol) rumen fluid, CRF2; 7.5% (vol/vol) each of mineral solutions 1 and 2; 0.5% (wt/vol) Trypticase; 0.05% (wt/vol) cysteine-hydrochloride; 0.06% (wt/vol) Na2CO; and 0.0001% (wt/vol) resazurin. Most carbohydrates were prepared as 10% wt/vol solutions, filter sterilized, equilibrated with CO2, and added (0.5% final concentration) aseptically to the basal medium. Medium and tests for esculin hydrolysis, gelatin liquefaction, nitrate reduction, and indole production were as described by Bryant and Doetsch (8). All of these media were prepared by the method of Bryant and Burkey (7) and tubed in 4-ml amounts in sterile, rubber-stoppered tubes (13 by 100 mm, Bellco Glass, Inc.) under 10% CO2-90% N. gas mixture. Growth, terminal pH, and biochemical tests were determined on cultures incubated for 7 days at 37 C. The inoculum medium in these studies was the same as M98-5 (Table 1) except glucose, cellobiose, and maltose were at 0.3% (wt/vol), and agar was omitted. All tests were inoculated with 3 drops of a 24-to 48-h inoculum medium culture under 10% CO2-90% N2 gas phase. Analysis of fermentation products. Volatile (acetic, propionic, butyric, and valeric) and nonvolatile (lactic and succinic) acids formed in glucosecontaining media were analyzed by gas chromatographic methods. The gas chromatograph used was a Hewlett-Packard model 5754B equipped with a flame ionization detector. Fermentation media samples (1 ml) were acidified with 0.1 ml of 6 N HCl, and any precipitate formed was removed by centrifugation. Acidified samples were analyzed for volatile acids on a column packed with 20% Carbowax 20 M TPA on Chromosorb W (Varian, 60 to 80 mesh, A/W, DMCS). Nonvolatile acids were methylated in acidified media and analyzed by a modification of the procedure of Hautala and Weaver (18). Formic acid was determined enzymatically by the method of Rabinowitz and Pricer (28). Hydrogen gas produced in gas glucose medium (12) was analyzed by gas chromatography on a Porapak Q (60 to 80 mesh, Applied Science) column and using a thermal conductivity detector. RESULTS AND DISCUSSION Effect of incubation time on colony counts with different roll-tube media. The data in Table 2 show the effect of incubation time on colony counts in 98-5, M98-5, and M10 media. Although the number of colonies continued to increase throughout the 14-day incubation period, the colony counts at 14 days were not significantly higher (P = 0.05) than those at 6 days with M98-5 and M10. In contrast, there was a significant (P = 0.05) increase in colony counts at each incubation time with 98-5. At all intervals of incubation, colony counts were significantly higher in M98-5 than in either 98-5 or M10 (Table 2, last column). Six-day colony counts in 98-5, M98-5, and M10 represented 79%, 92%, and 85%, respectively, of the 14-day counts. When 6-day counts were converted to percent recovery of the direct microscope cell count, 42.8% (98-5), 59.6% (M98-5), and 45.6% (M10) of the total bacterial population in cecal contents could be cultured. Deletion, addition, or substitution of components in M98-5 medium. Omission of rumen fluid from M98-5 reduced colony counts by 50%, whereas deletion of hemin alone made no significant difference in the number of colonies observed (Table 3). Colony counts in M98-5 lacking rumen fluid were similar to those obtained with M98-5 without rumen fluid and hemin. These results indicate that rumen fluid enhances the growth of many cecal bacteria when Trypticase is the only other source of organic growth factors with the exception of the carbohydrate energy sources. From the data, it is not possible to determine whether hemin is a nutritional requirement for cecal anaerobes because rumen fluid would be expected to contain heme (13). Preliminary nutritional studies on representative isolates (48 strains) from each group given in Table 6, however, indicate that few if any of these bacteria require hemin for growth or are stimulated by it. Hemin does augment the growth of most strains of Bacteroides isolated from the bovine rumen (10,13,22), human oral cavity, and intestine (16,24). Colony counts in medium M98-5 were not significantly (P = 0.05) altered when the following single changes were made (data not shown): (i) substitution of minerals S2 with minerals 1 and 2, (ii) deletion of Trypticase or substitution of Trypticase with Casamino Acids (0.2%), (iii) addition of yeast extract (0.05%), and (iv) deletion of glycerol. The reason for M98-5 yielding higher colony counts than 98-5 is not clear, particularly because deletion of Trypticase from M98-5 has no effect on colony counts. Perhaps the added energy source, maltose, or the combined addition of maltose, Trypticase, and glycerol in M98-5 enhances the growth of additional strains of cecal bacteria. Recent unpublished work by E. Barnes aMean colony counts times 1010 per gram, wet weight, of cecal contents obtained from six cecal samples after 6 days of incubation. Colony counts on all samples were made from a single batch of medium prepared with and without the noted deletions. b Means not followed by the same letter are significantly different at the 5% level of probability. chicken cecal anaerobes require liver extract to grow in carbohydrate-containing media. In our experiments, addition of liver extract (5 to 20%) to M98-5 (Table 4) did not significantly affect colony counts. To test the possibility that chicken cecal extract might replace rumen fluid for the growth of cecal bacteria, M98-5 (minus rumen fluid) was supplemented with 5 to 30% cecal extract. No increase in colony counts was noted (Table 4). Also supplementing M98-5 with varying amounts (2.5 to 20%) of cecal extract did not increase counts above the nonsupplemented M98-5 medium. Comparison of various media on percent recovery of anaerobes. In Table 5, the percent recovery of anaerobes in different nonselective media is compared. Although considerable variation (10 to 30%) was observed among cecal samples with all media tested, M98-5 medium gave consistently higher percent recoveries (mean of 60%) than M10, supplemented M10, or blood agar media. Barnes and Impey (3) have found that addition of liver and chicken fecal extracts to M10 is necessary for the isolation of many fastidious anaerobes from the chicken cecum. It may be concluded from our studies that supplementation of M10 with liver extract and/or chicken fecal or cecal extracts yielded 46 to 54% of the direct microscope cell count. These results indicate that such additions only marginally improve the recovery of bacteria b Means not followed by the same letter are significantly different at the 5% level of probability. from that of M10 alone. Moreover, we have consistently observed higher percent recoveries (15 to 25% higher) with M10 or supplemented M10 than those reported by Barnes and coworkers (3,4). With the blood agar roll-tube media tested, 25% of the microflora could be cultured. The high partial pressure exerted by CO2 (in the absence of added Na2CO0), however, altered the pH of VL blood agar to 6.5 and of Schaedler blood agar to 5.9 and, therefore, may have inhibited the growth of some bacteria. In subsequent experiments, six cecal samples were tested on VL blood agar and Schaedler blood agar buffered with Na2CO3 (0.4% vol/vol) to a pH of 6.8 to 6.9. The median percent recovery with VL blood agar was 33% (range 21 to 44%) and with Schaedler blood agar was 44% (range 33 to 52%) of the total microscope cell count. In contrast, the median percent recovery with M98-5 medium in these experiments was 81% (range 72 to 96%). These data indicate that for primary isolation of cecal anaerobes, prereduced blood agar media are inferior to rumen fluid media (M98-5). Also, adequate buffering of blood agar media in roll tubes is important when CO2 is used as a culture gas. In contrast to the culture counts in anaerobic roll tubes, plate counts of total aerobes on Eugonagar or of fungi on Sabouraud agar represented only 2% of the direct microscope counts. Most of the strains isolated on Eugonagar were identified as Escherichia coli and facultatively anaerobic streptococci in further studies. Characterization of cecal strains. From M98-5 medium, 298 strains of bacteria from six cecal samples were isolated. Strains were initially grouped according to a few morphological and physiological features, as well as fermentation products formed from glucose ( Table 6). Identification of selected strains was also based on a comparison of their reactions in various fermentation and biochemical tests to those of known species (Table 7). The predominant microflora is represented by at least 11 groups of bacteria, most of which are gram positive and strictly anaerobic, although two groups of facultatively anaerobic cocci were isolated. Group I was composed of gram-negative cocci or budding coci (1 by 1.5 to 2 Aim), which were clubor dumbbell-shaped cells arranged in pairs and chains. These organisms in a rumen fluid medium (Table 6) were obligately anaerobic and produced small amounts of H2 gas, and formic, acetic, propionic, and butyric acids from glucose. One strain (E10), further analyzed by the VPI Anaerobe Laboratory on peptone-yeast extract basal medium, was aReactions given are those for a majority of the strains within a group. Symbols: oxygen tolerance, A (anaerobic), F (facultative); +, positive reaction; -, negative reaction. Superscripts refer to reactions of a few strains; fermentation products from glucose: f (formic); A, a (acetic); P, p (propionic); b, (butyric); L, 1 (lactic); s (succinic). Upper-case letters refer to acids formed in amounts of 10 ,mol/ml of medium or greater, whereas lower-case letters refer to amounts less than 10 Mmol/ml. Products in parentheses are formed by a few strains. None of the strains tested digested cellulose, and only a few strains in Group VIb were motile. I11a IlIb Ilc IVa IVb IVc V VIa VIb 4 6 5 3 4 23 11 14 9 shown to ferment amygdalin, glucose, maltose, raffinose, and trehalose, to weakly ferment fructose, lactose, and salicin, and to hydrolyze esculin and produce formic and butyric acids from glucose. In preliminary nutritional studies, yeast extract was found to enhance the growth of bacteria in this group. A vitamin mixture containing thiamine-hydrochloride, nicotinamide, riboflavin, pyridoxine-hydrochloride, biotin, folic acid, and DL-thioctic acid could replace yeast extract as a growth stimulant. Foubert and Douglas (15), in a taxonomic analysis of anaerobic micrococci, described a strain (U5) that was isolated from the human uterus. This unnamed strain is similar morphologically and in fermentation characteristics (in peptone-yeast extract basal medium) to our Group I. Strains similar to Group I have also been isolated and described by Gossling (Abstr. Ann. Meet. Amer. Soc. Microbiol., p. 81, 1972) from human feces and by Barnes et al. (5) from chicken cecal contents. Group II was composed of facultatively anaerobic gram-positive cocci (1to 2-um diameter) occurring as singles, pairs, or tetrads. Many strains on initial isolation did not grow on aerobic plates. These strains fermented glucose, fructose, lactose, maltose, and sucrose, reduced nitrate, and formed lactic, acetic, butyric, and formic acids from glucose. They were catalasenegative and therefore differed from catalasepositive species of Micrococcus, Staphylococcus, and Sarcina (1). These organisms do not appear to belong to any previously described genus. Strains in Group III were represented by three different types of gram-positive cocci in chains. Group Ila was comprised of facultatively anaerobic cocci (1 to 1.5 by 1 to 2 ,um) in chains of lancet-shaped cells. None of the strains in Group HIa were hemolytic, but they fermented a wide variety of sugars and may be related to species of Group D streptococci (14). Some strains in this group did not produce an amount of lactic acid characteristic of fecal streptococci and, therefore, could not be species of the genus Streptococcus. Groups II1b and IlIc were similar in morphology and consisted of large gram-positive lancet cocci (1 to 1.5 by 1.5 to 2.5 Mm) arranged in pairs and chains. Similar fermentation products from glucose (acetic and small amounts of propionic and lactic acids) with large amounts of H2 gas were produced by strains of both groups. Group hIb produced H2S and fermented fructose and ribose, whereas Group Ec was weakly fermentative on most sugars. Yeast extract (Groups hIb and mc) and rumen fluid (Group IHb) stimulated the growth of these anaerobic streptococcal types. Groups 11Tb and mc represent species related to Peptostreptococcus Kluyver and van Niel according to emended descriptions of this genus by Rogosa (29). Group HIb may be similar to Hare Group VII isolated from human feces, but an insufficient number of characteristics were given in the publication of Thomas and Hare (33) for a suitable comparison. Strains in Groups hIb and Ec were not related to anaerobic streptococci isolated by Barnes and Impey (3) from poultry ceca or any of the several species of Peptostreptococcus classified by Holdeman and Moore (19) and may constitute new species of Peptostreptococcus. Group 1TIb strains also appear to be closely related (based on similar fermentation properties) to the chain-forming anaerobic streptococci (strain 21-29) isolated by Harrison and Hansen (17) from turkey cecal feces. Group IVa was one of the largest groups of anaerobes isolated and consisted of gram-positive, irregular, pleomorphic rods (1 by 2 to 3 Mlm). Most strains were strict anaerobes, but a few also grew aerobically. These organisms were identified as Propionibacterium acnes according to criteria of Holdeman and Moore (19). A number of variants were observed in this group that could be placed into biotypes A, D, and G as described by Pulverer and Ko (27). Barnes and Impey (4) have also isolated P. acnes from the chicken cecum. The growth of many of our P. acnes strains was enhanced by yeast extract, rumen fluid, and Tween 80. Group IVb consisted of some of the more active strains isolated. These were gram-positive organisms with rounded ends (1 by 2.5 to 4 Mim) occurring as singles, pairs, and long chains. Strains in this group were identified as Eubacterium rectale (19). Group IVb strains were also similar to strain EBG 1/80 isolated by Barnes and Impey (3) from chicken cecal contents. The growth of these strains was stimulated by yeast and cecal extracts and rumen fluid. Group IVc included gram-variable (many stained gram negative in older cultures) fusiform and lancet-shaped cells (1.5 by 2.5 Am) distributed as singles, pairs, and chains. The majority of strains in this group were nonfermentative, but a few hydrolyzed esculin and starch and fermented sucrose. They appear to be related to species of Eubacterium. Group V consisted of large, gram-negative pleomorphic, fusiform-shaped cells (1 to 1.5 by 2.5 to 5 Mm) in pairs and chains. These strains resembled species classified as Bacteroides clostridiiformis by Holdeman and Moore (19). They were characterized by production of lactic and acetic acids from glucose and by having a variable and limited fermentation capacity. Recently, known ATCC strains of B. clostridiiformis have been observed to form spores and are, therefore, species of Clostridium (W. E. C. Moore, personal communication). Spores have not been detected in our cultures of B. clostridiiformis even after prolonged incubation (3 weeks) at least in our rumen fluidglucose liquid medium. Similar strains of B. clostridiiformis were isolated from chicken cecal material by Barnes and Impey (3). Two types of spore-forming rods comprised Group VI. Group VIa contained pleomorphic, nonmotile cells (1 by 2 to 3 Mm) bearing terminal spores. These were similar to known strains of Clostridium ramosum in that glucose, fructose, lactose, maltose, mannose, melibiose, and sucrose were fermented (19). Strains in Group VIb were motile, fusiform rods (1 by 3 to 4 Mm) with subterminal spores and were species of Clostridium that could not be identified. Distribution of bacteria in the cecum. Results of this work indicate that 90% of all strains isolated from cecal material of six animals (5 weeks old) consist of at least 11 groups and subgroups of facultative and anaerobic bacteria (Tables 6 and 7). Approximately 10% of the strains were made up of unknown species (miscellaneous rods) that could not be identified. The predominant groups that were consistently isolated from high (10-9) dilutions of samples were distributed as follows: 7% as gram-negative budding cocci (Group I, unknown species); 12% as gram-positive facultative cocci (Group II, unknown species); 14% as streptococci (Groups IIIa, b, c; Streptococcus and Peptostreptococcus); 32% as gram-positive rods (Groups IVa, b, c; P. acnes, E. rectale and Eubacterium sp.); 14% as gram-negative rods VOL. 27,1974 68a005 (Group V, B. clostridiiformis); and 10% as spore-forming rods (Group VIa, b; C. ramosum and Clostridium sp.). Barnes and Impey (3), on the other hand, found that the anaerobic cecal microflora of the 5-week-old chicken was composed of 40% grampositive nonspore-forming rods and bifidobacteria, 40% gram-negative rods (Bacteroidaceae), and 15% strains of peptostreptococci and curved rods. Not only are there these differences in the distribution of bacterial types in our study and that of Barnes and Impey (3), but the latter investigators also isolated gram-negative rods such as B. fragilis and B. hypermegas. Moreover, bifidobacteria and lactobacilli, cultured from chicken intestinal contents by some investigators (26,30,34), were not recovered in any sample analyzed in this study, at least in high dilutions (10-9) of cecal material. We isolated similar strains of budding cocci, P. acnes and B. clostridiiformis, as did Barnes and co-workers (4,5). It may be concluded, therefore, that the relative proportion of the various bacterial groups within the intestinal microbial population of chickens are somewhat variable owing to such differences as breed, diet, growth conditions, and geographic location of animals. The relative proportion of bacterial groups would also be affected by the isolation methods used by different workers.

v3-fos

2018-04-03T02:05:23.668Z

1974-12-01T00:00:00.000Z

32273634

s2

Membrane concentration of infectious bovine rhinotracheitis virus from water. A membrane adsorption procedure was used to concentrate infectious bovine rhinotracheitis virus from 1-liter quantities of distilled water. Cellulose nitrate membrane filters (0.45-mum pore size) efficiently adsorbed this herpesvirus from water, and virus was recovered from the membrane by elution with 10 ml of fetal calf serum during sonic treatment. The average recovery rate was 70%. Infectious bovine rhinotracheitis (IBR) is a disease of cattle that produces upper respiratory inflammation as well as several other syndromes, the most economically important of these being abortion (6). Diagnostic investigations for a 4-month period in 1971 indicated that 67.8% of the 370 fetuses submitted to the North Dakota State Veterinary Diagnostic Laboratory were aborted due to IBR (3). The purpose of this investigation was to develop an IBR virus concentration procedure which could possibly be used to determine if this virus is present in livestock drinking water. The virus concentration methodology developed for this purpose was modified after numerous reported virus concentration techniques (1,4,5,8,9). (This study represents a portion of a thesis submitted by S. R. T. in partial fulfillment of the requirements for the M. S. degree in bacteriology at North Dakota State University, Fargo, 1974.) MATERIALS dispersed with 100 ml of trypsin (0.25%) containing 0.2% ethylenediaminetetraacetate, and the cell suspensions were centrifuged for 10 min at 500 x g to pellet the cells. The cells were then suspended in minimal essential medium containing 10% calf serum and antibiotics. The cells were cultured in disposable tissue culture dishes (60 by 15 mm) at 37 C in a CO2 incubator until confluent monolayers were formed. The minimal essential medium was then removed, and the monolayers were inoculated with 0.5-ml volumes of virus suspensions and the agar was overlaid (minimal essential medium with 1% ion agar). Upon development, plaques were resolved by staining the cells with a 1:1,500 neutral red solution. Membrane concentration procedure. One-milliliter volumes of an IBR virus suspension (4 x 104 PFU/ml) were diluted 1:1,000 in acetate-buffered (0.1 M, pH 6.5) water samples containing MgCI2 (0.5 M). The addition of MgCl2 was necessary to prevent virus loss during filtration (9). The samples were then filtered through type HA membrane filters (47-mm diameter, 0.45-,gm pore size; Millipore Corp., Bedford, Mass.) to adsorb the virus. Virus was released by suspending the filters in a 10-ml volume of fetal calf serum which was then sonically treated in an ice bath with the standard probe of a Biosonic IV sonic oscillator (Bronwill Scientific, Rochester, N.Y.) at maximum output. RESULTS AND DISCUSSION No IBR virus was detected in any of the type HA membrane filtrates, indicating complete virus retention. An increase in adsorption pH from 3.0, a recommended pH for the adsorption of enteroviruses (5), to 6.5 maintained the viability of this herpesvirus yet did not impede virus adsorption. Recovery of the adsorbed viruses was accomplished by sonically treating the membrane filters in 10 ml of fetal calf serum. To determine the amount of sonic treatment required for optimum IBR virus release, membranes were sonically treated for 2-to 5-min intervals. Maximum virus PFU were IBR VIRUS CONCENTRATION obtained after 3 min of sonic treatment ( Table 1). The inactivation of IBR virus was determined by sonically treating virus in fetal calf serum alone; virus was added to 10-ml samples and sonically treated for various times. The recovery rates were combined with the virus inactivation curve (Fig. 1) to indicate that, although 100% of the virus was released after 3 min of sonic treatment, 30% of the virus was inactivated. Although an average virus PFU recovery rate of 70% was obtained, the standard error of the mean was 9.4%. This variation within replicates indicates why there were no significant differences among mean recovery rates listed in Minutes of Sonication FIG. 1. Effects of increasing sonication on IBR virus recovery from type HA membrane filters suspended in 10 ml of fetal calf serum (0) and on the recovery of IBR viruses diluted in 10 ml of fetal calf serum (0). The membrane concentration technique was found more effective in recovering the IBR virus than were three other procedures (S. R. Tschider, M. S. thesis, North Dakota State Univ., Fargo, 1974). Polyelectrolyte 60 (11) and aluminum hydroxide (10) adsorption procedures were found to effectively remove virus from water samples, but the inability to recover IBR virus from these adsorbents restricted their usefulness. The aqueous polymer two-phase procedure that was utilized, a modification of the procedures of Shuval et al. (7), was found to be unacceptable. The two-phase virus concentrates were cytotoxic, and therefore the detection of virus PFU was not possible. All methods examined were modified from the originally reported procedures. Differences in virus characteristics (e.g., pH stability) between the enterovirus group and the herpesvirus necessitated these modifications. In summary, the membrane procedure was found to be an efficient method for obtaining 100-fold concentration of the IBR virus from water, with a recovery efficiency of 70%. Not only is this technique applicable to the concentration of IBR virus, but it also provides a procedure which could be used to concentrate other herpesviruses. Presently, this technique is being investigated for use in evaluating supplies of North Dakota livestock drinking water.

v3-fos

2017-08-27T20:19:14.640Z

1974-08-01T00:00:00.000Z

6614230

s2

Effect of Potassium Sorbate on Salmonellae , Staphylococcus aureus , Clostridium perfringens , and Clostridium botulinum in Cooked , Uncured Sausage Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate. Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate. Sorbic acid and its salts are used in many commercially prepared foods for preventing spoilage. Sorbic acid is selective in its antimicrobial activity. For example, it is a very effective inhibitor of yeasts in cucumber fermentations and yet permits the normal development of lactic acid-producing bacteria, except in those cases of high sorbic acid levels combined with a high initial brine content (3). Sorbic acid has been added to culture media for the selective isolation of catalase-negative lactobacilli and clostridia (5,7). All strains of Clostridium botulinum types A and B tested by York and Vaughn (8) were found to grow within 7 days at 35 C in beef liver infusion containing 3.0% sorbic acid. Hansen and Appelman (6) reported that sorbic acid neither inhibited nor stimulated the growth of C. botulinum in culture media. The observations that sorbic acid is selective in its antimicrobial activity and lack of inhibition for C. botulinum form the basis for a very restrictive use of sorbic acid and its salts in meat products. The only approved use consists of dipping the casings-for stuffed dry sausage. This application inhibits mold growth on the surface of the sausages during the long period they are held in drying rooms. Sorbate cannot be added to the meat portion of pizza pies even though it is permitted in the cheese and crust. Spoilage by molds results in significant loss of meat products at the retail and consumer levels. Addition of sorbates to the meat would reduce this loss of food. This study was designed to determine the effect of potassium sorbate on C. botulinum in cooked sausage in the event the product is temperature abused. Also, the effect of potassium sorbate on the growth of salmonellae, Staphylococcus aureus, and C. perfringens was examined. The methods of inoculating the product represent recontamination on the surface of the sausages after cooking (salmonellae and S. aureus) and spores surviving the cooking process (C. botulinum and C. perfringens). MATERIALS AND METHODS Meat. Cooked, skinless sausage links were used as the test system. The raw meat formula consisted of a mixture of beef and pork (87.95%), water (8.8%), sodium chloride (1.75%), and sugar and spices (1.5%). The fat content of the uncooked links was 43%. One lot of sausages was prepared in which 0.1% potassium sorbate (wt/wt; Mallinckrodt Chemical Works, Lodi, N.J.) was added to the ground meat formula prior to cooking. The links were heated to an internal temperature of 71 C and then frozen until needed. The cooked sausages had a pH of 6.2. The average weight of the links were 20 g each. Cultures. Five salmonellae cultures (Salmonella anatum, S. infantis, S. senftenberg, S. choleraesuis, and S. newport) were grown at 37 C in brain heart infusion broth (BHI; Difco). After incubation for 18 h, 262 on February 6, 2021 by guest http://aem.asm.org/ Downloaded from EFFECT OF POTASSIUM SORBATE ON SALMONELLAE the five strains were pooled for inoculation of the sausage. Five strains of S. aureus (S-6, 196E, 361, FDA 315, and ATCC 6538) were grown in BHI broth at 37 C and pooled after 18 h. Four of these strains are known to produce one or more of the known enterotoxins. Spores of three strains of C. perfringens (NCTC 8239, FDI, and ATCC 3624) were prepared as described by Duncan and Strong (4). The spore crops were harvested by centrifugation, washed several times in sterile distilled water, then resuspended in phosphate buffer (pH 7.0) and pooled to provide equal levels of each of the three strains for inoculation. Spores of five strains of type A (77A, 62A, 33A, 12885A, and 36A) and five strains of type B (9B, 40B, 41B, 51B, and 53B) C. botulinum were prepared as previously described (2). A pooled mixture of the 10 spore crops was used for inoculation of the sausage. Inoculation of meat. Our goal was to achieve an inoculum level of 1,000 C. botulinum and 1,000 C. perfringens spores per link and 1,000 salmonellae and 1,000 S. aureus per g of product. Sausages were inoculated on the outer surface with salmonellae by immersing the sausages in a dilute aqueous suspension of the pooled salmonellae. The sausages were removed from the suspension and allowed to drain. Each sausage link retained approximately 0.1 ml of the suspension and yielded an initial inoculum level of 32 to 38 per g or 640 to 760 per sausage link. The same method of inoculation for S. aureus yielded an initial level of less than 30 per g or less than 600 per sausage link. This inoculum level was below the limit of our method for enumerating S. aureus. The mixed spore suspension of C. botulinum was heat shocked at 80 C for 15 min and 0.1 ml was injected into the center of the sausage links. This yielded an inoculum level of more than 7,200 per g or 140,000 per sausage link, which was more than anticipated. The mixed spore suspension of C. perfringens was heat shocked at 70 C for 20 min before injection. The inoculum level was 320 per g or 6,400 per sausage link. The above methods were followed for product with and without potassium sorbate. Separate inoculum suspensions of salmonellae and S. aureus were used to dip sausages with and without sorbate. Storage and sampling of product. Samples representing each variable were packed separately in boxes (10 links per box) giving a net weight of about 200 g. Uninoculated control product was included for comparison. All product was stored at 27 C. Three boxes of each variable were removed for analysis at predetermined time intervals. A 90-g sample was removed from each box and blended in a Waring blender with 90 ml of sterile phosphate buffer (0.003 M, pH 7.0) for 1 min. After making appropriate dilutions, viable counts were determined. Total aerobic counts of uninoculated product were made by using standard plate count agar (Difco) with incubation at 27 C for 2 days. Salmonellae were enumerated by spreading onto brilliant green sulfa agar (Difco). Salmonellalike colonies were counted after 24 h at 37 C. Representative colonies were transferred into lysine iron agar slants (Difco) and examined for typical reac-tions. Samples inoculated with S. aureus were plated onto Baird-Parker agar (Difco) and examined after 24 h at 37 C. Samples inoculated with C. perfringens were plated into SFP agar (Difco), overlayed, and incubated in anaerobic jars at 37 C for 24 h. Counts were made and typical colonies were confirmed by transferring into nitrate-motility medium (1). Samples inoculated with C. botulinum were analyzed by a three-tube, most-probable-number technique using peptone colloid (Difco) modified by the addition of 0.1% dextrose, 0.05% ferrous sulfate, and 0.03% sodium thiosulfate. All tubes showing blackening after day 7 at 37 C were assumed to contain C. botulinum. Black tubes from the highest dilutions were selected at random and were confirmed by mouse test to contain botulinal toxin. Sausages inoculated with C. botulinum were also tested for toxin by centrifuging a portion of the 1: 1 suspension of the blended sample. Two white mice were injected with 0.5 ml of the supernatant fluid. A third mouse was injected with 0.5 ml of supernatant which had been boiled for 15 min. Death of the mice receiving the unheated extract and survival of the mouse injected with the boiled extract coupled with the C. botulinum viable counts were considered evidence for the presence of botulinal toxin. RESULTS AND DISCUSSION Potassium sorbate delayed the growth of the normal bacterial spoilage flora in the uninoculated product during the first day, after which growth was rapid ( Table 1). Growth of the salmonellae was markedy retarded by potassium sorbate. This agrees with a report (5) that sorbic acid inhibited salmonellae in culture media. Growth of S. aureus was inhibited during the first day, after which growth was rapid. C. perfringens declined to below detectable levels in all samples within the first day, independent of the presence or absence of potassium sorbate. Growth of C. botulinum was slower in the product containing potassium sorbate. Also, there was a delay in the development of botulinal toxin. Botulinal toxin was detected in 4 days in product without sorbate, but not until after 10 days in the product with sorbate. The product inoculated with C. botulinum had pH values of 6.4 and 7.1 after 4 days in samples with and without sorbate, respectively. A pH of 6.4 is sufficiently high to exclude the possibility that pH was a factor in the inhibition of botulinal growth and toxin production. These data demonstrate that the addition of 0.1% potassium sorbate to retard mold spoilage does not increase the public health hazard of cooked pork sausage in the event that it becomes temperature abused. To the contrary, the TOMPKIN ET AL. APPL. MICROBIOL.

v3-fos

2020-12-10T09:04:12.723Z

1974-03-01T00:00:00.000Z

237230290

s2

Enumeration of Food-Borne Clostridium perfringens in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar The SFP (Shahidi-Ferguson perfringens), TSC (tryptose-sulfite-cycloserine), EY (egg yolk)-free TSC, and OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agars have been tested for their suitability to enumerate Clostridium perfringens in naturally contaminated foods. Complete recoveries of C. perfringens were obtained in each of the four media, but only the TSC and EY-free TSC agars were sufficiently selective to ensure subsequent confirmatory tests without interference from facultative anaerobes. Because of some disadvantages associated with the use of egg yolk, EY-free TSC agar is recommended for enumeration of C. perfringens in foods. Several conditions for convenient shipment of foods and C. perfringens isolates with minimum loss of viability have been tested. The highest viable counts were preserved when foods were mixed 1:1 (wt/vol) with 20% glycerol and kept in a container with dry ice. Isolated C. perfringens strains remained viable for at least 2 weeks at ambient temperatures on blood agar slopes with a 2% agar overlay in screw-cap culture tubes. The presumptive enumeration of Clostridium perfringens is commonly based on the reduction of sulfite and the hydrolysis of lecithin in egg yolk media (10,11,22). In a recent publication (14), we listed a number of disadvantages associated with the use of egg yolk and showed that these could be overcome by a modified method. The method involves presumptive enumeration in pour plates with egg yolk (EY)-free agar containing 0.04% D-cycloserine (D-CS), tentatively designated EY-free TSC (tryptose-sulfite-cycloserine) agar, and stab-culturing black colonies in nitrate motility (NM) agar supplemented with glycerol and galactose. All of 71 strains of C. perfringens tested were recovered quantitatively in EY-free TSC agar, and all reduced nitrate to nitrite in supplemented NM agar. The present work was undertaken (i) to determine the applicability of EY-free TSC agar for the enumeration of C. perfringens in foods, and (ii) to evaluate conditions for the transport of foods and of C. perfringens isolates without undue loss of viability. MATERIALS AND METHODS Foods. Foods were purchased at local food service and retail establishments and abused to simulate conditions that have led to food-poisoning outbreaks in the past (2,5,23); i.e., they were handled with naturally soiled cutlery and incubated at temperatures conducive to growth of C. perfringens (Table 1). For incubation, the barbecued foods and roasts were wrapped in thin plastic film (saran) and enclosed in aluminum-lined food bags; the meat pie was kept in the original packaging (aluminum tray covered with plastic film); the other products were placed in sidearm flasks and deaerated with a water aspirator for 10 min. After incubation, food samples of 10 to 20 g were homogenized in a Waring blender with 0.1% peptone (9 ml per g of food) for 2 min at high speed. Ten-fold dilutions were made immediately with 0.1% peptone. Enumeration procedures. The following media were used for presumptive enumeration of C. perfringens: SFP (Shahidi-Ferguson perfringens) agar (22), TSC agar (11), EY-free TSC agar (14), and OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agar (9). They were prepared as described before (14). Volumes of 0.1 ml of diluted sample were spread on the surface of SFP and TSC agars, which were then poured over with 10 ml of cover agar (g2); the other two media were used in pour plates with 1.0 ml of diluted sample per plate. All plates were incubated anaerobically at 37 C for 20 h. For enumeration of C. perfringens spores, 10-ml samples of food homogenate at dilution 10-' in screw-cap test tubes (16 by 150 mm) were incubated for 20 min in a water bath at 75 C and cooled immediately in ice water. Confirmatory tests. Five or 10 presumptive C. perfringens colonies from each enumeration agar per food sample were stab inoculated into supplemented NM agar (14). For additional confirmation, colonies derived from food samples N to T (Table 1) and enumerated in EY-free TSC agar were also inoculated into lactose motility (LM) agar (22) and lactose Hemolysis. Five C. perfringens isolates from each of the food samples A to M were selected at random and streaked on sheep blood agar plates (Mogul Diagnostics, Madison, Wis.). Hemolysis was recorded after 24 h of anaerobic incubation at 37 C. Viability of C. perfringens in foods during storage. Food samples were finely chopped, mixed, distributed in amounts of about 10 g into tared 24-ml screw-cap vials, and weighed. The vials were stored at 4 and -18 C, and in a container with dry ice placed in a freezing compartment at -27 C to reduce sublimation of CO,. Within the dry-ice container, the vials were kept in small boxes with inside temperatures between -55 and -60 C, depending on the amount of dry ice. Some food samples were mixed 1:1 (wt/vol) with 20% glycerol (20) before storage. Viable counts were made in EY-free TSC agar after 0, 1, 2, 3, 4, 5, and 8 weeks of storage. Viability of C. perfringens on blood agar. Sixteen of the C. perfringens strains listed previously (14) were grown at 37 C for 20 h in 15 ml of cooked meat medium contained in screw-cap test tubes (20 by 150 mm). Each strain was transferred to 20 replicate blood agar slopes (Difco blood agar base with 5% defibrinated sheep blood) in screw-cap test tubes (16 by 150 mm). These were incubated at 37 C for 20 h. Ten slopes per strain were then poured over and completely covered with 2% agar (Difco) in distilled water. For each strain, groups of five identically treated tubes were stored at 4 C and at ambient temperature (21 to 24 C) for 1 to 8 weeks. After each storage period, single transfers were made from one tube of each group to cooked meat medium. Growth of C. perfringens at 37 C was recorded after 1 to 3 days. RESULTS Comparison of SFP, TSC, and EY-free TSC agars. Table 2 shows the C. perfringens counts for 13 foods (A to M). The data obtained with each of the three enumeration agars were almost identical. The C. perfringens counts for samples B, I, J, and L in SFP agar represent maximum counts because the large numbers of facultative anaerobes interfered in the confirmatory nitrite motility test. To ascertain that the recoveries were complete, we included SFP agar ( Table 2) and EY-free TSC agar (not shown) without antibiotics for foods A to H. Although most of these data represented maximum counts, they did not exceed those of the three selective media. In contrast to experiments with known strains (14), the C. perfringens colonies from nearly all the 13 foods produced opaque halos in both egg yolk media (SFP and TSC). Only 25 to 50% of the C. perfringens colonies from sample L had no discernible halos in either of the two media. In SFP agar, the egg yolk reaction of C. perfringens from sample B was completely masked by the large numbers of egg yolk-positive facultative anaerobes (Table 2). In both TSC agars (with or without egg yolk), the nonspecific counts (total counts minus confirmed C. perfringens counts) were either below the C. perfringens counts or were of the same order. In contrast, the nonspecific counts for some foods in SFP agar exceeded the C. perfringens counts by several logs. For samples B and H, the nonspecific counts were essentially as high in SFP agar as in the same agar without antibiotics ( Table 2). Since we counted on possible inhibition of C. perfringens in media with 400 ,ug of D-CS/ml, we also included EY-free TSC agar with 300 Mg of D-CS/ml in each experiment (not shown). At both D-CS concentrations, the C. perfringens and nonspecific counts were essentially the same, except for sample L where the nonspecific black-colony count was significantly higher at 300 Mg of D-CS/ml. Hemolysis. After enumeration in EY-free TSC agar and confirmation as C. perfringens by the nitrite motility test, five isolates per food were characterized further by hemolysis on sheep blood agar. Within each of 9 out of 13 groups, the 5 isolates all showed identical hemolytic patterns: they were all beta-hemolytic in 6 groups and partially hemolytic in 3 groups. Within each of these groups, the nitrite reac-tions were of similar intensities, but they varied between different groups. The isolates from theremaining 4 groups each showed two distinct hemolytic patterns. Viability of C. perfringens during storage. Table 3 shows the loss of viable C. perfringens in a number of foods after 1 week of storage at different temperatures. At 4 C, the log reduction varied from <0.2 (no detectable loss) to >3. Comparable losses occured at -18 C. In dry ice, the losses were considerably lower. Preliminary experiments indicated that the losses could be reduced further by storing the foods in 10% glycerol. This is confirmed by the results in Table 4, which show that the smallest loss in viable counts occurred when the foods were mixed 1:1 (wt/vol) with 20% glycerol and kept in dry ice. The log decreases in viable counts, plotted against storage time from 0 to 8 weeks, approached str'aight lines. We also tested the suitability of the common practice of shipping isolated C. perfringens strains on blood agar slopes. Table 5 shows that 'ND, Not determined. agars. To evaluate the suitability of the recently described OPSP agar (9) for enumeration of C. perfringens in foods, this medium was included in our viability studies. Table 6 compares the enumeration data obtained in EY-free TSC and OPSP agars for six foods that had been stored for 4 weeks in 10% glycerol at 4 C. The C. perfringens counts were essentially the same in the two agars, but the nonspecific counts were consistently higher in OPSP agar. Essentially the same results were obtained with foods stored at 4 C without glycerol (not shown). Confirmatory tests. Most of the black colonies derived from foods A to T and transferred to supplemented NM agar were confirmed as C. perfringens (1). However, plates of food samples J and L had black colonies that differed in appearance from the "typical" C. perfringens colonies; they could be counted and transferred separately and were all motile and negative for nitrite. Isolates derived from foods N to T were also tested in LM agar and lactose gelatin. All of 63 isolates identified as C. perfringens by the nitrite motility test fermented lactose rapidly and liquefied gelatin. Spores. The tests for C. perfringens spores in food samples N to T were all negative (<10 spores/g). Clostridial spores (4 x 103/g) were encountered in sample N, but these were all motile and did not produce nitrite. DISCUSSION In each of the four. enumeration agars (SFP, TSC, EY-free TSC, and OPSP), we obtained quantitative recoveries of C. perfringens for a Foods listed in Tables 1 and 4, enumerated after 4 weeks of storage in 10% glycerol at 4 C. each of the foods tested. Previous results with known C. perfringens strains were similar, except that the recoveries of some strains in OPSP agar were low (14). The facultative anaerobes were kept to relatively low numbers in both TSC agars but were much less inhibited in the SFP and OPSP agars. We previously listed a number of disadvantages associated with egg yolk agars for enumeration of C. perfringens. Since both the specific and nonspecific counts were the same for the two TSC agars, it appears that of the four media tested, the EY-free TSC agar is the most suitable one for enumeration of C. perfringens in foods. In 9 out of 13 foods, each of 5 C. perfringens isolates had the same hemolytic pattern. Although we did not determine the serotypes, this finding is consistent with reports of food-poisoning outbreaks (8,16,23) in which usually only one serotype of C. perfringens could be isolated from each incriminated food. Foods A and B ( Table 1) were halves of the same chicken, yet the five isolates from A were all beta-hemolytic, whereas those from B were partially hemolytic. This suggests that, of the factors determining which of the contaminating C. perfringens strains becomes predominant in a given food, the temperature is of particular importance. Duncan et al. (6) demonstrated that the synthesis of enterotoxin is associated with spore formation. Spores and enterotoxin, along with large numbers of vegetative cells, have been demonstrated in certain cooked meats (4,12,18). For some of these we have no information regarding their organoleptic qualities (18), but the others had offensive smells (12; F. M. Dework, personal communication). In contrast, cooked meats that are likely to be consumed seem to have either no or very few spores of C. perfringens (12,15). Spores Were also absent in contaminated foods tested in this work, although large numbers of C. perfringens cells were formed and some products were no longer palatable. These data are in accord with the concept (13) that the enterotoxin responsible for C. perfringens type A food poisoning is produced in vivo and that incriminated foods do not have to be assayed for the toxin in routine investigations of food-poisoning outbreaks. Investigators of C. perfringens outbreaks are often faced with the question of how to transport and store incriminated food samples and C. perfringens isolates, both without undue loss of viability (7). This work suggests that food samples may be suitably transported in mixture 1:1 (wt/vol) with 20% glycerol in a dry-ice container and stored in the same way or in a freezer at -60 C. Isolated strains may be trans-ported at ambient temperatures on blood agar slopes with a 2% agar overlay in screw-cap test tubes. As stated by Angelotti et al. (1), the only clostridial species listed in Bergey's Manual (7th ed., 1957) that is nonmotile and produces sulfide and nitrite is C. perfringens, with the possible exception of C. filiforme, which was isolated only in 1912 (3) and is no longer viable in its isolated form. However, other clostridia' with these three characteristics have been described: the unofficial species designated C. barati, C. perenne (17), Inflabilis barati, I. indolicus, and I. lacustris (21), and recent isolates from pheasant intestine (19). Some of these are likely to be identical. We recently isolated a clostridium with the same three properties from fecal specimens (manuscript to be published) that is distinctly different from C. perfringens and the clostridia listed above. These, as well as our fecal isolate and C. filiforme, may all be distinguished from C. perfringens by their inability to liquefy gelatin. According to Preivot (21), I. indolicus and I. lacustris may liquefy gelatin after prolonged incubation, but we found no liquefaction in lactose gelatin after 1 week of incubation with strains received from his laboratory, whereas C. perfringens strains liquefied gelatin consistently within 40 h, usually within 24 h, in this medium. In this work, all our nonmotile, sulfide, and nitrite-producing food isolates that were tested for liquefaction of gelatin were reconfirmed as C. perfringens.

v3-fos

2018-04-03T00:10:48.817Z

1974-06-01T00:00:00.000Z

10140486

s2

Volatile Flavor Compounds Produced by Molds of Aspergillus, Penicillium, and Fungi imperfecti Strains of molds Aspergillus niger, A. ochraceus, A. oryzae, A. parasiticus, Penicillium chrysogenum, P. citrinum, P. funiculosum, P. raistrickii, P. viridica-tum, Alternaria, Cephalosporium, and Fusarium sp. were grown on sterile coarse wheat meal at 26 to 28 C for 120 h. The volatiles from mature cultures were distilled at low temperature under reduced pressure. The distillates from traps -40 and -78 C were extracted with methylene chloride and subsequently concentrated. All the concentrates thus obtained were analyzed by gas-liquid chromatography, mass spectrometry, chemical reactions of functional groups, and olfactory evaluation. Six components detected in the culture distillates were identified positively: 3-methylbutanol, 3-octanone, 3-octanol, 1-octen-3-ol, 1-octanol, and 2-octen-1-ol. They represented 67 to 97% of all the volatiles oc- curring in the concentrated distillate. The following 14 components were identified tentatively: octane, isobutyl alcohol, butyl alcohol, butyl acetate, amyl acetate, octyl acetate, pyridine, hexanol, nonanone, dimethylpyrazine, tetra- methylpyrazine, benzaldehyde, propylbenzene, and phenethyl alcohol. Among the volatiles produced by molds, 1-octen-3-ol yielding a characteristic fungal odor was found predominant. In our previous paper (9) a description was presented of different volatiles produced by Aspergillus flavus. The purpose of this study was the identification of the volatiles produced by molds of the group Aspergillus, Penicillium, and Fungi imperfecti. MATERIALS AND METHODS Microorganisms. All the microorganisms studied were isolated from wheat grain and were maintained on Czapek-Dox agar slants at 3 C until used. Culture media and growth conditions. The culture medium used was coarse wheat meal sterilized at 1 atm for 45 min. The wheat meal was moistened to 60% water content and inoculated with conidia of pure culture suspended in physiological saline as described previously (9). After 3 to 4 days the culture medium (1 kg) was collected for the isolation step. Isolation of volatiles for the medium. The isolation of volatiles from the culture medium was carried out by vacuum distillation in an all-glass apparatus (10). The distillation step took 4 h and was done under nitrogen at 5 mm Hg. The temperature of the water bath was 35 C, whereas that of the cold traps in which the distillate was collected ranged from -10 to -80 C. The distillate collected in traps cooled to -40 and -80 C was extracted with CH2Cl2 and concentrated to a volume of 100 uliters (10). Gas chromatography. The separation of the volatile substances in the concentrated distillates was carried out with a Willy Giede model GCHF 18.3 gas chromatograph equipped with a flame ionization detector. The columns were stainless steel (3 m long; inner diameter 3 mm) packed with 15% Carbowax 20 M terminated with terephtalic acidon80to 100-meshacid-washed, dimethyldichlorosilane (DMCS)-treated Chromosorb W. Nitrogen was used as the carrier gas at a flow rate of 20 ml/min. Samples (2 gliters) were applied to the column which was held isothermally at 120 C. The percentage of individual volatiles was calculated from the peak area, the area of all the peaks on the chromatogram serving as the 100% value. Identification of the predominlant volatiles. The volatile compounds occurring in the extracts were separated on a gas-liquid chromatography column and then were identified by coincidence of relative retention times with those of known compounds, by mass spectrometry, chemical modification of the sample, and by olfactory evaluation. For identification purposes a combined apparatus gas chromatograph-mass spectrometer LKB 9000 was used. The columns were glass (3 m long, inner diameter 2.5 mm) packed with 10% Carbowax 20 M terminated with terephtalic acid on 80-to 100-mesh, acid-washed, DMCS-treated Chromosorb W. Helium, at a flow rate 10 to 20 cm3/min, served as the carrier gas. The columns were temperature programmed as follows: 5 C/min in the range 70 to 170 C. The ionization chamber was operated at 10' to 10' torr. A 70-eV source provided the mass spectra. Spectra were recorded from mass 30 to 250 in 100 s. Components were identified by comparing the mass spectra of the unknown to authentic compounds. The functional groups were identified also in the head space above the culture medium. The method of Hoff and Feit modified in this laboratory was used (11). The method is based on the reactions of functional groups of the volatile compounds with chemical reagents. The sensory evaluation was performed by a panel of 2 to 3 members by smelling the effluent from the column. RESULTS AND DISCUSSION The volatiles identified in the concentrated distillates from various strain cultures are listed in Table 1. 1-Octen-3-ol was found to be the main volatile component produced by all the molds studied. Its contents varied from 36.6 to 93.1% of the total volatiles. In this respect the most efficient appeared to be such molds as P. citrinum, P. raistrickii, Cephalosporium, P. funiculosum, and A. niger. Pure 1-octen-3-ol isolated from the molds by a gas chromatography trapping procedure exhibited a strong fungal resinous odor. In concentrations close to the threshold value its odor resembled that of the mushroom, Agaricus bisporus. According to the data presented in Table 1, the quantity of 1-octen-3-ol depends on the mold species and composition of the growth medium. Thus, A. ochraceus grown on such media as coarse wheat meal, starch, gluten, and plant oil was stated to produce volatile fractions differing both qualitatively and quantitatively. The contents of 1-octen-3-ol were calculated by measuring the peak area on chromatograms of the concentrated distillates. This strain was found to produce the highest amounts of 1-octen-3-ol when grown on coarse wheat meal (83.8%); lower yields were obtained on gluten (26.9%), starch (9.3%), and only traces on media containing plant oils. It should be stressed that the uninoculated coarse wheat meal contained only traces of the above mentioned compounds (9). A research was also carried out in this laboratory on the mushrooms: Agaricus bisporus and Boletus edulis (14,15). According to the data obtained, 1-octen-3-ol was the predominant volatile in Agaricus bisporus and B. edulis-78 and 82.5% of the total volatiles, respectively. It is noteworthy that this compound occurs in many other foods; its origin has not been established, however (1, 2, 4,5,7,8,12,13). According to the results of mass spectrometry and infrared analysis, 1-octen-3-ol produced by molds and mushrooms is identical (14,15). Thus, a number of strains can be used to produce foods showing the odor typical of mushrooms. In Table 2, data are presented on the contents of 1octen-3-ol in the head space above Agaricus bi- sporus and above the coarse wheat meal on which molds were cultivated. For A. oryzae, the vapors above the substrate contained 200 to 300 times lower quantities of 1-octen-3-ol as compared with those above the freshly harvested mushrooms, but more than in boiled ones. In the case of Cephalosporium, the level of 1-octen-3-ol in vapors was found to be only 2 to 3 times lower than in vapors above the fresh mushrooms. Certain mold species, for example A. oryze, are used in the production of soybean products (3,6). The mold species studied were found to be capable of producing other volatiles also (Table 1). Among the latter compounds, of special interest is 2-octen-1-ol, which yields a characteristic, unpleasant musty-oil odor. Some mold species, for example A. flavus (9) and A. parasiticus, produce this compound in large quanities. There are other mold strains, however, which produce 2-octen-1-ol in small amounts. Besides the compounds presented in Table 1, the following volatiles were identified tentatively by chemical reactions and mass spectrometry: octane, isobutyl alcohol, butyl alcohol, butyl acetate, octyl acetate, pyridine, hexanol, nonanone, dimethylpyrazine, benzaldehyde, propylbenzene, and phenethyl alcohol. Although the above compounds occur in low concentrations, they affect considerably the flavor of foods infected by molds. The volatiles produced by molds may be used as an index for the detection of food contamination by rapid instrumental methods.

v3-fos

2018-04-03T03:29:15.074Z

1974-07-01T00:00:00.000Z

6371346

s2

Fate of Ochratoxin A and Citrinin During Malting and Brewing Experiments The fate of ochratoxin A and citrinin during malting and brewing processes was studied by the use of naturally contaminated lots of barley, as well as by the addition of crystalline toxins to the mash. Complete degradation was observed for ochratoxin A from moderately contaminated barley lots and for citrinin added to mash. The use of highly contaminated barley resulted in transmission of ochratoxin A into the beer, but only 2 to 7% of the initial content was detected, corresponding to levels of 6 to 20 jg of ochratoxin A per liter of beer. Barley lots with this high ochratoxin contamination (1,000 to 5,000 Mg/kg) will be easily detected and, therefore, because of pronounced deterioration, should be rejected during inspection upon admittance to the breweries. Ochratoxin A and citrinin are mycotoxins with nephrotoxic properties, and they have been found as causal determinants in a naturally occurring kidney disease, porcine nephropathy (6). Kidney lesions have been induced experimentally in many animal species, and it is reasonable to expect the toxins to act on human kidneys as well. Ochratoxin A as a natural contaminant was first reported from the U.S.A. (12), where a sample of maize was found to be contaminated with approximately 150 Mg of ochratoxin A per kg. In inspection of maize from different regions of the U.S.A., ochratoxin A has repeatedly been found at levels of from 83 to 166 Mg/kg (13,14). This mycotoxin has also been detected in American barley samples (S. Nesheim, Annu. Meet. Ass. Offic. Anal. Chem., 85th, Washington, D.C., Abstr., 1971) at levels of 12 to 38 Mg/kg. Ochratoxin A was found in concentrations of 30 to 27,000 Mg/kg in 18 out of 29 samples of Canadian grain stored under damp conditions (11). The cereal samples consisted mainly of wheat but also included oats, barley, and rye. Contamination of Canadian wheat with ochratoxin A at levels of 20 to 100 Mg/kg was previously reported (10). Thirteen of the above mentioned samples of heating grain were simultaneously contaminated with citrinin at levels of 70 to 80,000 Mg/kg. The citrinin-contaminated samples included wheat, oats, barley, and rye. In Denmark, ochratoxin A-contaminated samples of barley and oats have frequently been observed at levels of 28 to 27,500 Mg/kg (6). Some of these samples were simultaneously contaminated with citrinin (160 to 2,000 Mg/kg). In Sweden, barley and oats have been found to be contaminated with ochratoxin A at levels of 16 to 410 Mg/kg (5). Most of the cereal samples reported here have been collected from lots which were intended to be used as animal feed. The breweries perform careful control of barley and other cereals before acceptance for malting and brewing purposes, because moldy cereals will spoil the flavor and other qualities of the beer, e.g., by causing gushing (3). In spite of the control, contaminated lots of cereals may pass, because organoleptical changes are minor and germination tests and microbiological control cannot prevent the passing of low-contaminated lots, which can only be detected by chemical analysis for mycotoxins. The fate of ochratoxin A and citrinin during the malting and brewing processes is therefore of considerable interest from a food hygiene and food safety point of view. (Some of the results presented here were included in a report at the 14th Congress of the European Brewery Convention, Salzburg, May 1973.) MATERIALS AND METHODS Barley. Lots naturally contaminated with ochratoxin A and/or citrinin were collected from farms, where the lots had caused kidney disease in pigs (6). Malting barley, of normal high quality, was obtained from the Carlsberg Breweries and used as uncontaminated barley (control). Malting and brewing. (i) Micromalting. Portions (70 g) of barley were steeped in 250-ml plastic beakers with 12 holes (2-mm diameter) in the bottom. Alternating periods of 8 h with water and 16 h without water were applied at 12 C. Steeping was finished when a moisture content of 43 to 44% was obtained, and germination was carried out in the same beakers at 12 C. Once a day the barley was taken out for inspection, and the kernels were loosened from each other. Total steeping and germination time was 9 days. The samples were kilned in nylon bags for 8 h at 45 C and 16 h at 75 C. (ii) Experimental brewing. Ten kilograms of malt was sprayed with 400 g of water and ground 10 min later. The mashing was an infusion system starting with 35 liters of water at 39 C. After 40 min, the temperature was raised to 51 C for 60 min, and 15 g of lactic acid was added. Eight minutes later, the mash was heated 1 C per min to 65 C, and after 40 min of saccharification the temperature was again raised in 1-C increments per min to 75 C and then held for 30 min. The mash was transferred to the lauter tun for filtration and sparging with 44 liters of water. The wort was boiled with hops for 90 min, cooled, and aerated. The wort was distributed into two vessels, each containing 25 liters of wort, and was fermented with 90 g of centrifuged yeast in each vessel at 10 C for about 7 days. After racking, the beer was stored at 5 C for 1 week and at 0 C for 4 weeks, after which it was filtered, carbonated, bottled, and pasteurized. (iii) Experimental brewing with barley and enzymes. Five kilograms of barley was steeped for 30 min at 50 C. The steep water was used later for mashing. After air-drying, the barley was ground and mashed in 20 liters of water at 50 C with addition of 5 g of bacterial amylase (BAN). The temperature was raised to 80 C in 60 min, and, after 30 min at this temperature, the barley-mash was pumped to the malt-mash prepared from 5 kg of ground malt and 17 liters of water at 20 C. To the mixed mash at 50 C was added 5 g of BAN and 5 g of bacterial proteinase (BPN). BAN (EC 3.2.1.1) and BPN (EC 3.4.4.16) were both obtained from Bacillus subtilis. The enzyme preparations were obtained from Novo, Copenhagen. After 30 min at 50 C, the temperature was raised in 1-C increments per min to 63 C, which was held for 45 min. Mashing was finished at 75 C, and the brewing was continued as described above. Germination of the barley was determined (2), and the following analyses were carried out on the wort. Extract concentration (% Plato) (1), attenuation (degree of fermentation) (1), nitrogen (7), viscosity by use of Hoppler viscosimeter, and color were measured in a spectrophotometer at 465 nm in a 1-cm cell. Mycotoxin analysis. (i) Solid samples. Samples (50 g) of solid material (barley, malt, spent grains) were ground and water was added and, after acidification, they were extracted with chloroform. Quantitation of citrinin (4) and ochratoxin (8) was carried out by the use of densitometer techniques. (ii) Liquid samples. Samples (350 ml) of liquid material (wort, beer) were acidified and extracted with chloroform. Quantitation was made as above. During the investigations, the following experiments were carried out: RESULTS Ochratoxin A. Moderately contaminated barley samples were used during experiment I, and, although the germination percentage of lot MT 480 was a little below the normal specification, a reasonable malting experiment could be performed. The results of ordinary malt analyses were normal, although there was a moldy smell during the malting, and no ochratoxin was detected in the malt (Table 1). Two heavily contaminated lots of barley were used for experiment II (experimental brewing). The lots had a moldy smell, very pronounced in MT 100, and because of a very low germination percentage, malting was out of the question, and brewing could be carried out only with addition of bacterial enzymes. As indicated in Table 2, there was a strong degradation of ochratoxin A during the brewing process, with the final beer containing 2 to 7% of the initial amount of ochratoxin A, resulting in a concentration of 11 to 20 gg/liter. Spent grains contained a proportional higher level, with a concentration of 80 to 210 ug/kg. The normal wort analyses (Table 3) showed an increase of soluble nitrogen and more pronounced color. The smell of the wort and the flavor of the beer was abnormal, especially in brew no. 1321. To show whether barley-malt enzymes had a different effect on the degradation than did bacterial enzymes, experiment III was conducted, with the addition to normal malt of crystalline ochratoxin A. A similar degradation, as in the previous experiment, was observed ( Table 2) with 4% of the initial amount found in the beer, equal to 6 gg/liter. On the thin-layer chromatography plates, fluorescent spots different from ochratoxin A and ochratoxin a were present in the wort but absent in the final beer. Citrinin. Malting and brewing experiments were intended to be done with the use of a naturally contaminated lot of barley. However, the germination percentage was zero, and no malting was possible. Instead, crystalline citrinin (10 mg) was added to normal malt (experiment IV), and no citrinin could be detected in the wort and spent grains. DISCUSSION The malting process completely degrades ochratoxin A present in moderately contami-nated barley lots. When heavily contaminated lots are used for mashing, a pronounced reduction of ochratoxin A takes place during the process, indicated by the presence in the wort of only 11 to 19% of the initial toxin content in the barley. The subsequent fermentation process degrades ochratoxin further, so that only 2 to 7% of the initial content is present in the beer, corresponding to 6 to 20 ug/liter. However, the barley used to produce this level in the beer was so heavily contaminated and deteriorated that similar lots would not be used by breweries. Citrinin degraded at an even faster rate than ochratoxin during the mashing process and was not present in detectable amounts in the wort. Only the use of highly contaminated lots of barley will result in production of ochratoxincontaminated beer. As these lots will be rejected during inspection upon admittance to the breweries, the problem of transmission of these mycotoxins from cereals to the beer seems very unlikely. The limitation of the present investigations is the use of TLC techniques only for toxic metabolite detection; no biological test systems for detection of possible toxic, nonfluorescent break-down products of ochratoxin A and citrinin were employed. LITERATURE

v3-fos

2019-03-21T13:11:43.974Z

1974-01-01T00:00:00.000Z

84453504

s2

Nutrition and feeding of African ungulates during quarantine at Dvur Kralove Zoo Ungulates brought to the Dvur Kralove Zoo after capture in Africa are put into quarantine for about three months. Immediately upon capture they are given feed similar to that which they will get later on in the zoo: African cattle pellets, bran, maize, high quality prcssed lucerne and twigs (4) . A quantity ofthis food accompanies the animals to the zoo, so that for a short period they continue with their African diet. In the course of their three months’ quarantine this is eventually completely replaced by locally available components. The accompanying tables indicate final rations at the end of the quarantine period. During quarantine itself the food constituents vary widely according to the animal’s ability to adapt itself to the local diet and according to its general state of health. These rations are supplied in winter, in summer hay being replaced by mixtures of lucerne and corn, and frozen twigs by frcsh ones. While the animals are quarantined we provide only winter rations, even though the quarantine pcriod usually takcs place in suniiner. This is mainly becausc fresh fodder brings the danger of flatulence, and also because the animals adjust more easily to hay than to green mixtures, Some smaller and more delicate species have to be taught to eat zoo food, especially pellets, by initially substituting dried apples, dried beet pulp, acacia husks imported direct from Africa etc. Larger species usually acccpt their new diet willingly from the vcry beginning. W e try to maintain the health of animals in quarantine at the same level as it was 011 arrival, and gradually to improve it, even at the risk of making them overweight. Only after they are accustomed to the local diet, and at the end of the quarantine period do we slowly reduce the quantity of grain foddcr. Grain fodder also partially replaces twigs, which are supplied in greater quantities in the quarantine period. Later on, twigs are given only as a supplement to hay and green feed. Current research has shown that ruminants may be divided into several groups according to their food requirements (3) : course of their three months' quarantine this is eventually completely replaced by locally available components. The accompanying tables indicate final rations at the end of the quarantine period. During quarantine itself the food constituents vary widely according to the animal's ability to adapt itself to the local diet and according to its general state of health. These rations are supplied in winter, in summer hay being replaced by mixtures of lucerne and corn, and frozen twigs by frcsh ones. While the animals are quarantined we provide only winter rations, even though the quarantine pcriod usually takcs place in suniiner. This is mainly becausc fresh fodder brings the danger of flatulence, and also because the animals adjust more easily to hay than to green mixtures, Some smaller and more delicate species have to be taught to eat zoo food, especially pellets, by initially substituting dried apples, dried beet pulp, acacia husks imported direct from Africa etc. Larger species usually acccpt their new diet willingly from the vcry beginning. W e try to maintain the health of animals in quarantine at the same level as it was 011 arrival, and gradually to improve it, even at the risk of making them overweight. Only after they are accustomed to the local diet, and at the end of the quarantine period do we slowly reduce the quantity of grain foddcr. Grain fodder also partially replaces twigs, which are supplied in greater quantities in the quarantine period. Later on, twigs are given only as a supplement to hay and green feed. Current research has shown that ruminants may be divided into several groups according to their food requirements (3) : I . Species feeding on rough common grass, eating the upper parts which contain more grass leaves. This group includes hartebeest Alcelaphus, Hunter's antelope Dntnalisn~s hunteri, blesbok D. dorcus phillipsi and bontebok D. dorcas dorcas. 2. Species requiring fresh grass and fresh water. These include waterbuck Kobus ell&siprymnus and K. megaceros, White-bearded gnu Connochacta taurinus albojinbattrs and Cape buffalo syncerus cafer. 3 . Species, such as Oryx, which feed on dry grass, including the stalks. Animals beIonging to these three groups consume about 95% grass and only about 5% of other herbs, including shrubs and leaves. Species requiring a juicy and concentrated diet constitute another broad grouping : Knowledge of food composition from the analysis of stoiiiacli contcnts of captured animals has helped us greatly in preparing their diet during the acclimatisation period and while they adapt to local food. Table I shows rations as provided for ungulates on transfer from quarantine to their permanent living quarters. The most difficult to accliniatise and adapt to a new diet are hartebeest and springbok which have only light protein requirements. W c gradually wean them from lucerne to hay. They arc content: with rough, mountain hay, but it must be well dried without trace of fungi or moulds to which they react adversely. They are very fond of oat straw and, most of all, of a mixture of husks and corn, but this must be fully ripe and tlie mixture pressed into bales. In suinnier, also, they prefer very ripc mixtures. If the components are not sufficiently ripe, we have to add hay and straw in the ratio of z : I . Generally speaking, these species are very difficult to adapt to the local hay, husks and corn fodder. The Huntcr's antelope in particular, at first refused totally to acccpt thc local feed until we attached small bundles of foddcr vertically to their feeding troughs. The grain acccptcd most willingly was crushed oats. Later on oat flakcs proved to be better for themindeed, some authors believe that crushed oats arc unsuitable for smaller animals, which digcst them with difficulty or not at all. Tlicre also exists the danger of injury to the mucous nieiiibranes of the mouth, or of inflaniiation causcd by oat chaff. Thc spccies most adaptable to a captive diet proved to be the Cape buffalo, which eats any sort of hay, cven of poorer quality, and green fodder. Like all our zoo animals they become acciistonicd to pellets only slowly, but accepted carrots in any quantity. White-bearded giiu, waterbuck and similar species also adapt casily to our diet. Oryx, like hartebeest, at first preferred straw, even of poorer quality, to hay and got used to hay only after it had been mixed equally with straw. In summer they also prefer riper mixtures with a larger proportion of cereals than husks. They accepted grain and carrots readily from the very beginning, so that it was not necessary to introduce them to dried apples. Giraffe and Greater kudu also fed well from the first. During quarantine we try to give them as many twigs and leaves as possible, although later on twigs serve merely to supplement the hay and green feed. In suininer they get whole leafy branchesfrom which they will strip even the bark -while in winter we give them dried or frozen twigs taken from the tree tops. They prefer sweet hay with a high proportion of dried leafy plants and mixtures with a high husk content. Lesser kudu, on the other hand, are relatively difficult to adapt. They prefer lucerne to hay, at first accepting very little grain foddcr and no carrots at all. Even after three months they did not accept all the food they should have. Roan antelope, too, were reluctant to adapt to hay and green fodder, even when mixed with straw, and this situation improved only gradually. Grain, however, was accepted readily from the very beginning. Eland, despite their natural preference for leaves and twigs, adjusted to hay and green feed without trouble. All these animals are given vitamin and mineral supplements in gradually increasing quantities from the start of quarantine. C O N CL U S I 0 N S Whereas for grazing species crudc protein intake represents about 8-10% of dry inattcr, it is higher for animals which feed triainly on leavesabout 11-14%. In the lattcr case, to raise the protein level of their dict at thc beginning of quarantmc, we added low fat dried milk powder to tlie animals' grain fodder. At London Zoo, for examplc, young deer arc given r3-r60/: crude protein, and adults 11-12?>. Bilby (2) and Abranis ( I ) suggcst 20% for young aninials and IO'~" for adult?. The level of bcta-cxoteli as a food coinponc11t Table 2 proved sufficient for all animals. At the end of the quarantine period, animals were on the whole very healthy, food intake being quite sufficient both in quantity and quality. It is most important, however, to observe absolute hygiene in the preparation of all food, as the animals, as yet unused to a ZOO diet, are very sensitive to the smallest impurity. Acclimatised specimens are no longer so delicate. Feeding techniques also play an important role. Very often subterfuges have to be employed to make the animals accept the unaccustomed food.

v3-fos

2019-03-19T13:14:43.742Z

1974-12-01T00:00:00.000Z

237231442

s2

Further Characterization of Tissue Distribution and Metabolism of [14C]Aflatoxin B1 in Chickens The distribution and metabolism of [14C]aflatoxin B1 in chicken tissues were further investigated. Previously dried and frozen ethyl acetate extracts of liver, heart, gizzard, breast, leg, blood, and fecal samples were obtained from either layer or broiler chickens fed subclinical levels of [14C]aflatoxin B1. Treatment of these extracts with either carboxypeptidase A, leucine aminopeptidase, pepsin, or trypsin revealed that an average of 50% of the 14C detected in the acetate extracts was a liberated peptide (or amino acid) conjugate of [14C]aflatoxin B2a. When a prepared standard of B2a was made by incubation of B1 with cold dilute aqueous HCl, the Rf values and absorbance maxima were identical with those of the tissue extracts after enzymatic treatment. the tissue extracts after enzymatic treatment. Aflatoxins, secondary metabolites produced by certain strains of Aspergillus, are recognized as food and feed contaminants having worldwide significance. The ubitiquous nature of these organisms suggests a potential hazard as far as human and animal intoxications are concerned. Results of earlier research (1)(2)(3)(4)14) have indicated that there was no transfer of aflatoxin to edible tissues of chicken fed diets containing crude aflatoxins. However, we have recently reported the distribution and metabolism of [PC ]aflatoxin B1 in broiler (8) and layer (9) chickens. In these studies, we found that both broilers and layers excreted approximately 90% of the`4C administered by crop intubation daily for 14 days. Significant amounts of "C were detected in the blood, liver, heart, gizzard, breast, leg, and feces. Treatment of aqueous extracts for conjugated steroids by treatment with beta-glucuronidase revealed that approximately 32% of the "C detected in the aqueous extract was a liberated glucuronide conjugate of [14C]aflatoxin M1, with the remaining '4C being uncharacterized. Aflatoxin M1 was also recently found to be the major metabolite in studies of the excretion and metabolism of orally administered aflatoxin B1 in rhesus monkeys (5 Extraction and characterization of fractions. After incubation, each fraction was extracted with 3 x 15 ml of chloroform. The chloroform extracts were then dried under nitrogen and stored at 5 C. The dried material was redissolved in 1.0 ml of absolute methanol and 10 Mliters spotted on TLC plates and developed along with the B,. standard. Blue fluorescing spots corresponding to the standard were scribed and scraped from the plates, eluted with chloroform, dried, and R, values and absorbance Inaxima in absolute methanol were determined. Afterward, 0.5 ml of the methanol solutions were reduced to dryness in liquid scintillation counting vials and the radioactivity was assayed according to the procedure outlined by Mabee and Chipley (8). Characterization of [("CJB,, recovered from excreta, organs, and tissues. Enzymatic treatment of ethyl acetate fractions isolated from broiler and layer chickens with the enzymes listed above, followed by chloroform extraction, revealed that an average of 50% of the total radioactivity observed in the ethyl acetate fractions before enzyme treatment was now located in the chlorofrom extract. Values of about 40% liberation for the more specific peptide-hydrolyzing enzymes, carboxypeptidase A and leucine aminopeptidase, and 55% liberation for the less specific enzymes, pepsin and trypsin, were obtained for all of the excreta, organs, tissues, and blood fractions tested in the present study. TLC of chloroform extracts yielded faint bluish spots with Rf values of 0.18 to 0.20. When these spots were removed from TLC plates and concentrated, their absorbance maxima were identical to those described for B2a by Dutton and Heathcote (6). Subsequent liquid scintillation spectrometry of the eluted fluorescing spots revealed that approximately 90'% of the radioactivity observed in the chloroform extract was confined to the isolated fluorescing material. A summary of the total distribution of radioactivity reported earlier for broiler (8) and layer (9) chickens, as well as of the results of the present study, is presented in Fig. 1. DISCUSSION The results obtained in the present study indicate that both broiler and layer chickens can metabolize the majority of aflatoxin B, when administered at relatively low levels. Aflatoxin conjugates are the predominating form of metabolite produced. Absorbance maxima, R, values, and radioactive-fluorescing spots on TLC are suggestive evidence that ["ICJaflatoxin B, is metabolized to a major extent (see Fig. 1) to a peptide (or amino acid) conjugate of B2a and to a lesser extent to a glucuronide conjugate of Ml. These metabolites are soluble in aqueous (sodium acetate) extracts. They were found in pooled samples of each of the biological tissues, organs, and excreta that were assayed in approximately the same ratios as shown in Fig. 1. Patterson and Roberts (11) reported that livers of chicks, guinea pigs, and mice metabolized aflatoxin B, into small amounts (5 to 10%) of M,, whereas the major metabolite (90%) was the aflatoxin hemiacetal B2a. Patterson Mabee and Chipley (8,9). 7,1972) has also shown that B2a, which is relatively harmless when taken orally, was an acute hepatotoxin whenever formed from B, by liver microsomal enzymes. Pohland et al. (13) investigated the acid-catalyzed addition of water to the vinyl ether double bond of aflatoxin B1. The hemiacetal (B.a) produced was biologically inactive to chicken embryos and tissue cultures at concentrations substantially higher than the minimal lethal dose of B. These authors also found that B. was highly unstable, probably as a result of its existence in a phenolate form. Patterson and Roberts (12) reported that a nicotinamide adenine dinucleotide phosphate reduced form-linked cytoplasmic enzyme system of duck liver reduced aflatoxin B1 to the cyclopentenol, aflatoxicol. Isolated liver microsomes were found to contain an enzyme that hydrates the vinyl ether double bond of aflatoxin to form its hemiacetal (B2a). They further stated that yields of hemiacetal were difficult to assess because of strong protein (or amino acid) binding, which probably involves the formation of Schiff bases with free amino groups. Aflatoxin itself and aflatoxicol reportedly were bound to protein (or amino acids) less readily than was the hemiacetal. This would account for the conjugated metabolites found in the presently reported study. Implications. The conjugated metabolites reported in this study are examples of the "detoxication" process described by Harper (7). In this process, toxic substances (aflatoxin B1) are converted into nontoxic forms (aflatoxin conjugates of Bla and M1) that are most efficiently removed by excretory routes by tissues. He also stated that conjugation might be accomplished by the combination of the metabolite to a variety of compounds including amino acids, glucuronic acid, "active" sulfate, and acetate. This appears to be the case in the present study. Conjugated aflatoxins can be liberated by animal systems in the presence of the appropriate enzyme. Since aflatoxins M1 (8,9) and B2a were successfully liberated from conjugates with liver, stomach, and pancreatic enzymes in vitro, a similar reaction could take place in tissues of animals administered aflatoxin conjugates. The liberated or unconjugated aflatoxin would probably then undergo reconjugation as a part of the detoxication process in an animal's system, resulting in possible deposition in the animal's tissues. In a recent report, Patterson (10) has reviewed the role of metabolism as a factor in determining the toxic action of aflatoxins in different animal species. He stated that once toxin has entered liver cells, the agency causing tissue injury in a particular animal species is dictated by the rate and pattern of aflatoxin metabolism. When it is metabolized slowly, untransformed toxin is probably the active molecular species with chronic liver damage the probable result. When it is metabolized rapidly, metabolites rather than original toxin appear to be involved. He also reported that acute liver damage may be caused by the intracellular formation of aflatoxin hemiacetal (B2a) in many species. Once again, the results of the present experiment indicate that classical nonpolar extraction procedures cannot be relied upon for the isolation and identification of aflatoxins in animal tissues.

v3-fos

2018-04-03T00:45:31.613Z

1974-04-01T00:00:00.000Z

26679352

s2

Microbiology of the Frankfurter Process: Salmonella and Natural Aerobic Flora Salmonella senftenberg 775W added to frankfurter emulsion was killed during normal processing in the smoke house when internal product temperature was 71.1 C (160 F) or above. The thermal destruction point of S. senftenberg 775W in frankfurters (temperature at which no viable cells were detected) was a function of the length of time of the process rather than of the starting number of cells. Heating of frankfurters to 73.9 C (165 F) substantially reduced the total non-salmonella count. For total non-salmonella bacterial flora and salmonella, relatively little thermal destruction occurred below 43.3 C (110 F). The heating step can bring about a 7-log cycle decrease (108 to 101/g) of bacteria present in the raw emulsion. The flora of this high-bacteriological-count raw emulsion was predominantly gram-negative rods. Variation in the number of bacteria (both total and salmonella) surviving at various temperatures during processing was attributed to slight variations in the temperature pattern of the smoke house during its operation. An integration process was devised which allowed calcula-tion of exposure to temperatures above 110 F (43.3 C) on the basis of degree-minutes. Plots of degree-minutes versus log of surviving bacteria were linear. The salmonella plot had a greater slope than that of the total non-salmonella flora, indicating that salmonellae are more heat sensitive than the bacterial population as a whole. The predominant bacteria surviving the heating step were micrococci. These micrococci were able to increase in number in or on the frankfurters during storage at 5 C. Salmonella senftenberg 775W added to frankfurter emulsion was killed during normal processing in the smoke house when internal product temperature was 71.1 C (160 F) or above. The thermal destruction point of S. senftenberg 775W in frankfurters (temperature at which no viable cells were detected) was a function of the length of time of the process rather than of the starting number of cells. Heating of frankfurters to 73.9 C (165 F) substantially reduced the total non-salmonella count. For total non-salmonella bacterial flora and salmonella, relatively little thermal destruction occurred below 43.3 C (110 F). The heating step can bring about a 7-log cycle decrease (108 to 101/g) of bacteria present in the raw emulsion. The flora of this high-bacteriological-count raw emulsion was predominantly gram-negative rods. Variation in the number of bacteria (both total and salmonella) surviving at various temperatures during processing was attributed to slight variations in the temperature pattern of the smoke house during its operation. An integration process was devised which allowed calculation of exposure to temperatures above 110 F (43.3 C) on the basis of degreeminutes. Plots of degree-minutes versus log of surviving bacteria were linear. The salmonella plot had a greater slope than that of the total non-salmonella flora, indicating that salmonellae are more heat sensitive than the bacterial population as a whole. The predominant bacteria surviving the heating step were micrococci. These micrococci were able to increase in number in or on the frankfurters during storage at 5 C. Although salmonellae are often found on the raw meats used to manufacture processed meat products (14,15) and in fresh pork sausage (7,12), they are rarely found in the processed product (14,15). Apparently salmonellae do not survive the heat processing given these products. Few data are available which detail the effects of actual processing conditions on the destruction or survival of salmonellae or of the aerobic (non-salmonella) bacterial flora in processed meat products. Furthermore, information about the flora that survive heat processing could be useful in predicting the shelf life of the products as well as the adequacy of the heating step of the process. Therefore, we investigated the thermal destruction of: (i) the total (nonsalmonellae) bacterial flora, and (ii) salmonellae during the heating step of frankfurter processing. Salmonella senftenberg 775W, the most heatresistant strain known (11,16), was chosen for the main part of this study. We thought that if S. senftenberg 775W were killed during the normal heating step, all other serotypes which might be present in raw frankfurters also would be killed. The types of bacteria surviving the various temperatures during frankfurter processing were studied in terms of their potentials for spoilage. MICROBIOLOGY OF THE FRANKFURTER PROCESS and 0.529 g of sodium ascorbate per kg of meat and fat. All batches contained (per kilogram of meat and fat): 19.8 g of sugar, 25.1 g of NaCl, and 5.29 g of commercial spice mixture. Emulsions were prepared in a Koch model 25 high-speed Schnellkutter; all components were added to the cutter bowl, with the curing agents were added as a solution in 100 ml of water. The mixture was chopped until the temperature of the emulsion was 15.6 C. The emulsion was stuffed in 23-mm No-Jax casings (Union Carbide) and linked. The linked frankfurters were cooked in an air-conditioned smoke house operated according to the following schedule: 10 min at 64.4 C dry bulb (DB), 30 min at 62.8 C DB and 57.2 C wet bulb (WB), 45 min at 73.9 DB and 60 C WB, and finally 87.8 C DB and 76.7 C WB until the internal temperature was 73.9 C (71.1 C for the salmonella experiments). Frankfurter temperature was monitored continuously during heating by a thermocouple inserted into a raw frankfurter and was recorded by an Electronik model 16 recorder (Honeywell). For experiments where frankfurters heated to different internal temperatures were to be analyzed bacteriologically, short strands of linked frankfurters were prepared and then removed from the smoke house as the product reached selected temperatures. These frankfurters were then immersed in an icewater slurry to cool rapidly. Microbiology: general. A 50-g amount of raw emulsion or frankfurter cooked to specific temperatures was aseptically removed from the casing and blended with 200 ml of 0.1% peptone water for 1 min at high speed in a Waring blender. Appropriate dilutions were pour-plated with tryptic soy agar (Difco); 0.1% peptone water was used as diluent. Colonies were counted after 4 days of incubation at 25 C. Gram stains of the various colony types were examined with a microscope; the catalase test was also run on these same colonies. The term total flora refers to the total, aerobic, non-salmonella flora counted by this procedure. Preparation of salmonellae-containing frankfurters. Two methods were developed for inoculating the raw emulsion with salmonellae. In the first, the bag method, raw emulsion was added to a plastic bag and an appropriate number of salmonellae suspended in green food dye-peptone water were added and thoroughly mixed with the emulsion. In the second, the cutter method, the diluted salmonellae in green food dye were added directly to the Schnellkutter bowl before the emulsion was formed; thus, the organism became an integral part of the emulsion network. The green food dye was used as a tracer to ensure uniform mixing of the contaminant and as a deterrent to unauthorized consumption of the finished product. Microbiology: salmonella. Raw, salmonella-containing emulsions or salmonella-contaminated frankfurters cooked to specific internal temperatures (120 g) were aseptically removed from the casings and blended with a mixture of 400 ml of selenite cystine broth (Difco) and 2.4 ml of Tergitol 7 for 3 min at high speed in a Waring blender. Both the total direct and most probable number (MPN) salmonella counts (three tubes, three dilutions) were made from this blended emulsion (BE). For the direct viable salmonella count, 0.1 ml of the BE, or a dilution thereof, was spread on the surface of brilliant green agar plates (BGA; Difco), and typical magenta colonies were counted after 24 h at 37 C. Colonies on BGA were routinely subjected to serological analysis; Proteus spp. or other bacteria which might give colonies similar in appearance to salmonella were never observed. For the MPN salmonella counts, 431-, 43.1-, and 4.31-g portions (equivalent to 100, 10, and 1 g, respectively, of original frankfurter emulsion) of BE were removed, placed in appropriate containers, and incubated for 24 h at 37 C. Samples from these enrichments were then streaked onto BGA plates and incubated for 24 h at 37 C. Typical magenta colonies were picked and inoculated into lysine-iron agar (Difco); the salmonella were finally confirmed by serology with 0 antisera. The MPN per gram was then calculated by use of an MPN table. The direct salmonella method was used for frankfurters and raw emulsion containing relatively high levels of salmonellae, whereas the MPN method was used when low levels of salmonellae were anticipated. S. senftenberg 775W and S. dublin were from the laboratory stock collection and were grown for 24 h in tryptic soy broth (Difco) incubated at 37 C. A 0.5-ml volume of culture was added to 100 ml of 0.1% peptone water containing 5 ml of a green food dye (see above); 10 ml of this diluted culture-food dye mixture was used to inoculate 1 kg of raw emulsion. This dilution gave an approximate count between 10' and 105 organisms per gram. Processing equipment was cleaned and sanitized as follows. Solid, raw emulsion was removed, placed in a container, and autoclaved. The equipment then was soaked and washed in a solution of Chloroterg cleaner (Oakite), followed by rinsing and steam treatment. Throughout the processing operation, laboratory personnel wore disposable plastic gloves, aprons, and lab coats which were autoclaved after use. The green food dye in the emulsion also facilitated clean-up by permitting location of any contaminated emulsion. RESULTS 'l'he importance of thermal destruction of bacteria during frankfurter processing cannot be overemphasized. Because of this importance, the bacteriology of the process was studied in detail with regard to both total flora and to salmonella. Figure la shows the variation in temperature response of frankfurters during four experiments (I to IV) using the same smoke house schedules. The causes of the difference might be external conditions of temperature, humidity, etc., or just the normal variation of the smoke house; these could not be controlled more precisely under our experimental conditions. Although all batches of frankfurters were prepared from the same starting meats that had been frozen, and the frankfurters in the four experiments were heated according to the same smoke house schedule, viable cell counts varied considerably, especially the final counts. In experiment III, the surviving bacteria were gram-positive, catalase-positive, spore-forming rods (bacilli), whereas in the other three experiments the survivors were gram-positive, catalase-positive cocci (micrococci). Among the three experiments, the number of micrococci surviving also varied. In general, most of the killing occurred after the product temperature reached 43.3 C (110 F). At the beginning of heating, the predominant flora were gram-negative rods with some micrococci; most of the gram-negative rods were destroyed by the time the product temperature reached 60 C. Above 60 C, only micrococci were found. Data from three experiments with S. senftenberg 775W are presented. Although the salmonellae-containing frankfurters were processed according to the same smoke house schedule, considerable variation in the time required to reach various internal temperatures was observed. These smoke house runs were conveniently designated as slow-heating (experiment VII), medium-heating (experiment V), and fastheating (experiment VI) houses. Figure 2a shows viable salmonella and product temperature plots for experiment V; in this experiment, salmonellae were added to the emulsion by the bag method. No viable salmonellae were found in products heated to 68.3 C and above; relatively little thermal destruction occurred before 57.2 C. Figure 2b shows viable salmonellae and product temperatures for experiment VI; in this experiment, salmonellae were added to the emulsion by the bag and cutter methods. Viable salmonellae are detected in the product heated to 68.3 C (0.018 organisms per g with the bag method and 0.056 organisms per g with the cutter method). The apparent discrepancy between the salmonella thermal death point in experiments V and VI may be explained by their respective heating curves. In experiment V, the product took 35 min to go from 65.6 to 68.3 C, whereas in experiment VI the product took only 4 min. Thus, in experiment V, the product was in this lethal temperature increment longer, and complete destruction occurred. Although similar numbers of salmonellae were added in experiment VI, there was a log cycle difference in the number of salmonella found at zero time between emulsions inoculated by the bag and by the cutter method. Possibly the high shearing forces of the cutter destroyed some salmonellae. We had thought that incorporation of the salmonellae into the emulsion might protect the organism during heating. However, after the initial kill during incorporation into the emulsion, the curves were essentially parallel. The apparent discrepancy at 68.3 C probably reflects the difficulties associated with quantitating small numbers of salmonellae. The cutter method simulates commercial operations. Figure 2c shows viable salmonellae and product temperatures for experiment VII. The salmonellae were incorporated by the bag method. In this experiment, no viable salmonellae were detected in products heated to 65.6 C. Comparison of the heating curve with those of experiments V and VI indicates that, in experiment VII, the product was heated more slowly than in the previous two experiments. The differences in times required to reach various internal temperatures and the viable counts at these temperatures are presented in Table 1. Some of the differences in the counts may be attributed to differences in the starting counts. However, examination of the time required for the product to reach the temperatures indicated that, in experiment VII, the product reached temperature slowly, whereas in experiment VI the product reached temperature rapidly, and experiment V was intermediate. The viable salmonella counts are reflected in the different times required to reach temperature (Table 1). In the "slowest experiment," VII, no viable salmonella were detected in product heated to 65.6 C, whereas in the fastest experiment, V, the last temperature at which viable salmonellae were detected was 68.3 C. The above data on the thermal destruction of total flora and added salmonellae further support what is already known: thermal destruction of bacteria is a time-temperature function. Therefore, a method was sought which would combine the lethal effects of time and temperature on bacterial destruction during frankfurter processing. Since Fig. 1 and 2 indicate little, if any, thermal destruction below 110 F (43.3 C), only temperatures above this were considered. The method devised was an integration process accomplished by tracing the area of the recorder chart within the lines formed by the reference temperature (ice bath) and the product temperature. For the time parameters, the base lines were from the time the product temperature reached 110 F (43.3 C) (taken as zero) and up to the time the product took to reach various temperatures. The weights (in grams) of the areas of paper bound by these parameters were converted into arbitrary units which were plotted against log survivors for total flora and for salmonella; 125 degree-minutes equal 0.03288 units (grams). (Fig. 3). Best-fit straight lines and correlation coefficients were calculated by linear regression. The correlation coefficient was -0.77 for total flora, based on collective data from the four separate experiments, and was -0.95 for salmonellae, based on collective data from the three separate experiments. Furthermore, the slope of the lines indicated that the total flora (slope = -0.89) are much more heat resistant than the salmonellae (slope = -5.45). This was not unexpected if one considers that: (i) heat completely destroyed the salmonellae, and (ii) the total flora represented a mixture of bacteria with different heat resistances (gram-negative rods and gram-positive cocci with a few gram-positive spore-formihg rods) and that the normal frankfurter heating process destroys completely only the gramnegative rods (organisms of relatively low heat resistance). The data employed to prepare Fig. 3 and also Fig. 5 were subjected to further statistical analysis using the least-squares method. In all instances, the second-degree, or quadratic, component was not significant. This provides additional support for the validity of our integration method for computing lethality, and thus a linear relationship between log number and degree-minutes is the valid one. The bacterial contributions of the various components of frankfurter emulsion to the bacteriological load of raw emulsion were determined ( Table 2). The count for finished raw emulsion was lower than the sum of the bacterial load contributed by the components. The count of finished raw emulsion was 7.0 x 10O organisms per g, whereas the load contributed by the components in the proportion found in Apparently some bacteria were killed in the Schnellkutter during emulsion formation. This decrease in total was less than the approximately 1-log cycle decrease of salmonellae in the Schnellkutter. To compare the bacteriological quality of our finished emulsion and finished frankfurters with those produced commercially, a count was made on a sample of commercial frankfurter emulsion. It had a count of 3.2 x 105 organisms per g and contained micrococci and gram-negative rods. We then processed frankfurters from this emulsion and made a bacteriological count of them. The major difference between frankfurters we processed and those processed commercially appeared to be the surviving flora. The commercially processed frankfurters had a count of 2.6 x 102 organisms per g and contained gram-positive, sporeforming rods; in contrast, ours had a count of 1.2 x 102/g and contained micrococci and some gram-positive rods (nonsporeforming). Data from our own and commercially processed emulsions and presented by Heiszler et al. (8) indicate that only 2-to 3-log cycles of killing occur during normal frankfurter processing, e.g., the counts decrease from 105 organisms per g in the raw emulsion to 102/g in the finished frankfurter. Whether this represents a dependence upon the flora present or simply the ability of the process to kill only this number of bacteria was investigated. After partial thawing, beef, pork, and pork fat were ground through a 3A6-inch plate and then stored for 6 days at 5 C. Bacteriological counts made during this aging period indicated that at 6 days the counts were still increasing. These aged meats had a marked putrid odor and could not be employed to manufacture frankfurters in any plant under federal inspection (Mandatory Meat Inspection, U.S. Code of Federal Regulations, Title 9, revised 1973, p. 337-338). These aged meats and fat then were made into frankfurters and processed in the smoke house. Samples were removed as various internal temperatures were reached during the heating; these samples were then analyzed bacteriologically ( Fig. 4 and 5). Because of the extremely poor quality of the starting meats, these frankfurters were not tasted. Although the starting count of the raw emulsion was 3-log cycles higher than in any previous experiment, the bacterial count of the finished frankfurters processed to 73.9 C was 3.7 x 10'/g (Fig. 4). Thus, the process is capable of destroying large numbers of bacteria. A plot of degree- minutes versus log survivors is given in Fig. 5. Examination of Gram stains of bacteria surviving various temperatures showed that, at temperatures up to 62.8 C, gram-negative rods and micrococci were present; at 65.6 C and above, only micrococci were found. Linear regression analysis of the data gave a correlation coefficient of -0.9660 and a slope of -2.55. Although the added salmonellae were killed by the heating step of frankfurter processing ( Table 1), mishandling of the raw emulsion before processing might allow proliferation of the salmonellae to such high levels that they might not all be killed by the heating step. To investigate this possibility, we inoculated raw emulsion with salmonellae and incubated it at different temperatures. Viable salmonellae were counted after 24 h. In most instances, there was neither appreciable growth nor death ( Table 3). The presence or absence of nitrite seemed to have no effect. In contrast to salmonellae, the total flora of raw emulsion increased markedly during incubation at 20 and 35 C ( Table 3). frankfurters indicated that very low numbers of bacteria survive the heating step. Yet, frankfurters spoil. A question was then posed: Can the bacteria which survive the heating step increase in number during subsequent low temperature storage and ultimately cause spoilage? Unpeeled frankfurters were stored in plastic bags at 5 C and examined bacteriologically at intervals. This method of storing frankfurters was chosen to eliminate the possible introduction of potential spoilage bacteria during peeling, handling, and packaging. The storage data (Table 4) indicated that the increase in count during storage is essentially due to growth of bacteria on the surface of the frankfurter. Since the frankfurters were not peeled until analysis, the bacteria/yeast counted should represent only those which survived the process and grew. The yeast probably was present in very low numbers after processing and only after 14 days of storage grew out and became the dominant flora. DISCUSSION Heating and smoking of raw emulsion during frankfurter processing serves several functions: (i) sets the emulsion and forms the skin of skinless frankfurters, (ii) kills the trichinae, (iii) accelerates cured meat color development, (iv) imparts a desirable smoky flavor to the frankfurters, and (v) decreases the bacterial content of the frankfurters. The first function required a heating schedule as given above. The other functions occur simultaneously with the first. In terms of shelf life extension (keeping quality) and public health, the last function is perhaps the most important for the finished product. The normal heating given frankfurters both destroyed the salmonellae and reduced the total flora. Thus, the product was made safe from a public health view and the shelf life should be extended. Because of the uniquely high heat resistance of S. senftenberg 775W (11,16), our results can be extended to include all known serotypes. Thus, any processor following a heating schedule similar to ours for cooking frankfurters to 71.1 C internal temperature should produce salmonella-free frankfurters. Our data support the observation (14,15) that salmonellae are not associated with processed frankfurters and form the basis of our conclusion: salmonellae do not survive the heating step of the frankfurter process. The presence of salmonellae in finished frankfurters would indicate either underprocessing or recontamination after processing. Whether our data for salmonellae in frankfurters can be extended to include other processed meat products is not known, but they should at least include bolognas, which are similar in composition and processing schedule to frankfurters, as well as other luncheon meats. Our data also indicate that heat resistance of salmonellae is not dependent upon the method of contaminating the emulsion (bag versus cutter method). Incorporation of the organism into the emulsion (Fig. 2b) offered no protection to the salmonellae during the heating. Bayne (3), in his study of the heat resistance of S. typhimurium in ground chicken, found that the organism suspended in emulsified fat was destroyed as readily as when it was suspended in meat; our data for S. senftenberg 775W support this. Bayne et al. (4) found that the heat resistance of salmonella in ground chicken muscle was similar to its heat resistance in other foods. Thus, heat resistance is not product dependent. Beloian and Schlosser (5) found for various baked foods that heating to 71.1 C (thermocouple in the slowest heating region of the food) was sufficient to destroy S. senftenberg 775W added to these foods. Dawson (6), in reviewing thermal destruction of salmonellae and other pathogens in turkey rolls and other meats, recommended an internal temperature of 71.1 C (at the cold-est point) for destruction of low levels of pathogens in these products. The inability of S. dublin and S. senftenberg 775W to grow in raw frankfurter emulsion (Table 4) is in contrast to an observation of Jensen (9) for S. aerotrycke (S. typhimurium). He observed that S. aerotrycke had a 4-h lag in stuffed, raw frankfurters incubated at 28.9 C. Since he presented no actual data, the extent of growth can not be ascertained. The cause for the difference between our work and that of Jensen is not known; it could be related to differences between strains of salmonellae, differences in preparation of emulsion, etc. He observed no lag with the total aerobic bacteria incubated at 28.9 C. Our experiment with raw emulsion having a high initial bacterial count showed that the heating step of the frankfurter process can decrease bacterial numbers by 7-log cycles when the predominant flora was gram-negative rods (pseudomonad types). Whether the heating step could cause a similar decrease if the flora was completely micrococci or gram-positive, nonsporeforming rods is not known. However, these types might not be expected in meat held at 5 C for 6 days. Warnecke et al. (13) found at least a 3-to 6-log decrease in counts of viable bacteria in bologna processed to an internal temperature of 68 C. They did not list the types of bacteria found in the raw emulsion or the finished bolognas, but judging from the amount of kill observed in their experiment, the starting flora was probably gram-negative rods. Examination of the bacteria surviving the heating step of our process indicated an almost exclusive flora of micrococci. Upon storage at 5 C, these survivors increased in number. Although our frankfurters held unpeeled for up to 21 days showed no signs of spoilage (slime formation, off odor, discoloration), micrococci have been shown to cause spoilage in packaged frankfurters (2). Probably, however, they would have spoiled the product after further storage. Our studies indicated that bacteria which survive the heating step can grow at 5 C and ultimately could cause spoilage. The yeast probably survived in such low numbers as to be undetectable immediately after processing. However, after destruction of the competing flora and in the selective environment between the frankfurter skin and the casing, certain numbers of the surviving flora can grow and cause spoilage. Further support for bacteria surviving the heating step as the causative agent for spoilage comes from the observation that, for frankfurt-ers stored 21 days, the surface count is very close to the total count (Table 4). At zero time, the surface of a frankfurter was almost sterile ( <5 organisms/g). The selective environment of the casing permitted extensive growth of certain surviving organisms. When frankfurters made from high-bacterialcount raw emulsion were stored unpeeled at 5 C, the flora and count increase were similar to frankfurters made from normal count emulsion. Thus, initial count of emulsion apparently had no effect on the keeping quality of the finished frankfurters. One of the most important functions of the heating step of the frankfurter process appears to be destruction of any salmonellae present and reduction of the total bacterial count. Heating of the frankfurters during normal processing to an internal temperature of 71.1 C provided at least a 2.8 C (5 F) margin of safety for destruction of salmonellae and reduced substantially the total bacterial count. This recommended final temperature of 160 F (71.1 C) for frankfurters is commensurate with the final processing temperatures of 68 to 72 C (154 to 162 F) used in commercial practice (10). The heating step destroyed very large numbers of gram-negative rods when they were present in the raw emulsion. Variation in the numbers of salmonellae and total flora that survived at different temperatures was explained by calculating exposure to temperatures above 110 F (43.3 C) on the basis of degree-minutes. Plots of degree-minutes (in arbitrary units) versus log survivors gave straight lines. The predominant organisms surviving the heating step were micrococci which, in our experiments, increased to substantial numbers on the product during storage at 5 C.

v3-fos

2018-04-03T02:18:00.480Z

1974-08-01T00:00:00.000Z

33119219

s2

Quantitative infrared photoanalysis of selected bacteria. A technique to measure transmitted infrared radiation from minute biological systems is described. Infrared color film was exposed by radiation transmitted through bacterial colonies. The resultant photographic image was unique for each species of bacteria examined and spectral analysis of the image provided differential light emission patterns which could be quantitated. A formula for developing numerical comparisons among bacterial colonies was provided. The results of this numerical procedure gave quantitative relationships for the total infrared data from each microbial colony and made possible the differential identification of ten species of medically significant bacteria. A technique to measure transmitted infrared radiation from minute biological systems is described. Infrared color film was exposed by radiation transmitted through bacterial colonies. The resultant photographic image was unique for each species of bacteria examined and spectral analysis of the image provided differential light emission patterns which could be quantitated. A formula for developing numerical comparisons among bacterial colonies was provided. The results of this numerical procedure gave quantitative relationships for the total infrared data from each microbial colony and made possible the differential identification of ten species of medically significant bacteria. Of all differences among bacterial species, none are more generally relied on for identification than cellular morphology and metabolism (2). These characteristics are demonstrated through variations of colony color, texture, density, geometry, rate of growth, gas evolution, heat liberation, and chemical composition. The collective effect of these features produces unique patterns of absorption or reflectance in response to interaction with the electromagnetic spectrum (3,4). A number of studies (8,9) have shown that infrared spectrophotometry can be used to distinguish between various genera of bacteria. However, these procedures are time consuming and demand specialized equipment not readily available in nonresearch and clinical laboratories. This paper describes results of an infrared analysis of bacterial colonies which may have future-application in the clinical laboratory. MATERIALS AND METHODS Organisms and media. Bacteria from the Brigham Young University culture collection were maintained on agar slants consisting of 12% beef heart infusion (Difco), 1.2% peptone, 1.2% gelatin, 0.6% dextrose, 0.6% casein, 0.5% disodium phosphate, 0.4% sodium citrate, and 0.9% agar. The transparent medium used to obtain transmitted infrared (TIR) data consisted of 1.6% nutrient agar, 0.25% yeast extract (Difco), and 0.25% glucose. A 20-ml amount of medium was placed into disposable petri dishes (100 by 15 mm). The medium was inoculated with approximately 50 bacterial cells of the desired strain, and the cells were distributed evenly over the agar surface. All cultures were incubated at 37 C. Infrared photomicrography. Figure 1 is a sche-matic diagram of the instrumentation used for infrared recording of bacterial colonies. A Leitz Photolab II photomicroscope was fitted with a 35-mm camera back and further modified as follows. (i) Light eminating from the tungsten source was enclosed in a light-tight corridor to the microscope stage ( Fig. 1, item E). (ii) The stage was modified such that light filters could be imposed between the light and the object (Fig. 1, item D). A Leitz brightfield microscope condenser was used with both aperture diaphrams wide open. One eyepiece was fitted with a micrometer for centering the object and measuring bacterial colonies under examination. A Kodak (Rochester) number 87 infrared filter was positioned in the light path between the source and the object (Fig. 1, item D). This filter allows transmission of radiation from 720 nm to beyond 1,500 nm. Power to the light source was controlled by using a filament transformer equipped with an ampmeter and voltmeter. Ektachrome infrared film (Kodak) having a single emulsion series number was stored at 4 C in moisture-tight containers prior to use. Infrared recording procedures. IR film to be standardized was allowed to come to room temperature (22 C) before placing it in the camera back. The power source was equilibrated and a petri dish containing culture media was placed on the microscope stage. The microscope was focused on the agar surface through the camera window. The power was adjusted to 1 A and the film was exposed by using an A.S.A. of 100 and a shutter speed of 1/8 s. Subsequent exposures were made at 0.25 A power increments up to 8 A. The film was then developed and used to demonstrate film characteristics (indicated in Fig. 3, 4, and 5). Each subsequent role of film was compared to this standard to insure proper control of exposure and development procedures. Each roll of film must be standardized to correct for variation in the media and for photographic procedures. This standardization is accomplished by re-205 moving the number 87 filter and focusing the optics on the agar surface. The filter is replaced and two frames of film are exposed, one at 3.5 W of power and the other at 8 W. When these control frames have been exposed the filter is again removed and the bacterial colony to be analyzed is brought into focus under the microscope. The filter is replaced and final focusing is done through the camera by using the colony edge for proper planar adjustment. Because colony morphological detail is not required, it is unnecessary to make focal length adjustment for changes in colony height. The power is adjusted to a value between 6.5 and 8.0 W to obtain proper exposure of the film, and the colony is photographed. Development of the film is done immediately after exposure. Exposed IR film was developed by using Kodak E-4 processing (6). Fresh Kodak developer solutions were used to develop only four rolls of film and then were discarded. Constant film-chemical agitation was maintained during the development procedures. Recommended film development times and water bath temperatures were held constant (7). A 10-mm wide strip was cut from the center of the photograph of the desired colony. This picture was then attached to a 35-mm photographic film leader 10 cm long and 10 mm wide. The sample and leader were then threaded into the scanning assembly of a Beckman DBG spectrophotometer (Fig. 2). The spectrophotometer was fitted with a 25-cm chart recorder set to linear response. With the light wavelength set at 580 nm and by using a beam width of 1.5 mm, the sample was passed in front of the light beam at 2.5 cm/min. The density-geometry pattern of the photographed colony was recorded on the chart paper. For infrared analysis, the scanner as shown in Fig with the light wavelength set at 760 nm, the wavelength scan is activated and completed at a wavelength of 380 nm. This procedure is, in principle, a comparison between two infrared (IR) photographs of identical agar surfaces, one of which includes a bacterial colony. The difference in appearance, as seen be the IR film, provides a means for characterization of the colony. This difference is measured by using a scanning spectrophotometer to reveal subtle changes in the layers of dye on the film. RESULTS Kodak Ektachrome IR film contains three layers of visible and infrared-sensitive film dyes. Exposure results in red coloration as a function of the incident infrared intensity. Figure 3 shows analysis of a plain transparent agar plate photographed at two infrared intensities. The data from such samples are in TO response to the transmitted IR which has only been attenuated by the transparent agar. These data serve as standards for peak positions and 60 intensities which can then be used for analytical 2 comparisons. Two characteristics result from analysis of these recordings: (i) peak area, which is designated as the static infrared unit (SIRU) and is determined by dividing the area 40 under the peak (Fig. 3, peaks 1 emulsions and experimental conditions and should be obtained for each roll of film that is used. Should a recording be made at a variety of infrared intensities on a particular roll of film, any standard SIRU value can be found from this data. As each roll is its own reference, the significance of the control standard is to give an indication of how far from an optimal reproduction any particular film roll is. Data obtained by this technique of transmitted IR recording provides two types of information about the bacterial colonies. (i) a spectrophotometric scan of the photographed bacterial colony gives information which is highly characteristic for many species. (ii) Analysis of the photographs of bacterial colonies gives an indication of the alterations in the IR radiation that is absorbed by the colonies as compared to the unattenuated IR radiation of an appropriate agar standard. Fig. 6 and 7A) result from differences in colony geometric configuration and are obtained by passing the film through the spectrophotometer operating at the indicated wavelength. The transmission recordings ( Fig. 6 and 7B) are obtained by keeping the film stationary in the spectrophotometer and scanning the photograph over a range of wavelengths of light. In these figures it should be kept in mind that the peak areas represent the greatest cell density in the colonies. The recorded IR pattern in these figures is mediated by colony composition as it interacts with radiation from the illuminating source. Figure 8 shows the changes in infrared transmissions of bacterial colonies as a function of incubation time. The change in infrared transmission with age indicates change in geometry, and composition which collectively affect the colony's ability to absorb, transmit, and reflect IR radiation. However, such transmission change was not obvious for all of the organisms tested. Much of the final characteristic of the transmission patterns was not developed until late in the colony growth process. It was also I .;e. The transmission and geometric patterns provide three characteristics which are used to determine quantitative values: (i) geometric conformation ( Fig. 6 and 7A); (ii) the SIRU Fig. 6 and 7B); and (iii) peak positions ( Fig. 6 and 7B). Using the values from these three characteristics it is possible to construct a table showing quantitative relationships for each species of organism examined (Table 1). In Table 1, two characteristics of the infrared transmission recording are combined into one value called beta. The position of peaks 1 and 2 as well as the SIRU for each peak is determined from the recorded transmission curve (Fig. 6 and 7B). The values thus obtained are used in the following formula to obtain beta. , = (xs) /(ty) where ft equals beta value; x equals (SIRU of peak 2 from the standard) + (wavelength of maximum transmission of peak 2 from the standard); s equals (SIRU of peak 2 from the test organism) + (wavelength of maximum transmission of peak 2 from the test organism); t equals (SIRU of peak 1 from the test organism) + (wavelength of maximum transmission of peak 1 from the test organism); and y equals (SIRU of peak 1 from the standard) + (wavelength of maximum transmission of peak 1 from the standard). The structure of the equation was derived in its present form as a result of attempts to determine the ratio most useful in indicating maximum differences between sample values and suggests no other relationships. The base of the peak in millimeters produced by a 580-nm scan of a microbial colony photograph (see Fig. 6A). b Alpha: First number represents the number of transmission peaks obtained on photoanalysis (refer to Fig. 6A), the slant has no mathematical meaning, whereas the second number equals the base times the area of the peak divided by 100. c Peaks 1 and 2 refer to the transmission recording peaks (see Fig. 6 and 7B). d SIRU: static infrared unit; A: wavelength of peak transmission. "Beta: a value derived according to the formula on page 209. ' SD, standard deviation of beta from five experimental measurements. DISCUSSION The data presented in this study have demonstrated that bacterial colonies, even of closely related species, possess sufficient structural and compositional differences to provide characteristic IR interactions. These characteristics can be recorded on IR-sensitive film. Subsequent spectrophotometric analysis of the film provides data useful in quantitating specific differences. The colony's infrared transmission response suggests a dependence on the collective effect of colony density, color, geometry, and chemical composition. IR transmitted through the bacterial colonies has the capacity to reduce dyes in the film which are IR wavelength specific. The reduction of these selective dyes is characteristic of the colony through which the IR radiation has passed. The data presented in this paper are easily obtained, require limited specialized equipment, and may be feasible in a clinical as well as research setting. Control experiments using white light and noninfrared Ektachrome film have failed to produce similar geometric data or light interaction data which could allow bacterial identification (Fig. 9). The overall effect of the inherent bacterial IR emissions (passive infrared) incident on the film is difficult to appraise. E. coli produces about 1.4 x 10-5 W of IR per s per colony area when ideal emissivity conditions exist (1, 3), whereas Staphylococcus aureus produces only about 9.0 x 10-6 W of IR per colony area per s. However, currently available IR film requires a minimum of about 3.0 x 10 2 W of IR per colony area per s, in order for the film to be exposed sufficiently to be detected by photoanalysis (4,5). Therefore, the inherent IR of the colonies is not sufficient to effect a response on the film due to their low power and the long wavelength of such passive emissions. These limitations in detection of passive bacterial IR require that a carrier infrared source be supplied, such as the photomicrographic system used in these studies. Due to the low sensitivity of the film and short IR wavelength required for exposure, it is doubtful that the small but characteristic amount of passive infrared emission from the bacterial colonies contributes significantly to the overall transmission response of the test organisms. The data contained in Table 1 do not suggest that any interpolations can be made regarding specific information on chemical composition of the cells. Variations in the system such as film emulsion changes, changes in film developing chemicals, compositional changes in bacterial growth media, and different microphotographic systems prevent the data in Table 1 from being established as standard values for the organisms shown. With future refinement and standardization of media and film development, it may well be possible to establish a reference by which unknown organisms could be identified by using this infrared transmission procedure.

v3-fos

2018-04-03T02:26:26.169Z

1974-04-01T00:00:00.000Z

33575789

s2

Requirement for and Sensitivity to Lysozyme by Clostridium perfringens Spores Heated at Ultrahigh Temperatures' The requirement of ultrahigh temperature (UHT)-treated Clostridium perfringens spores for lysozyme and the sensitivity of heated and unheated spores to lysozyme were studied. The UHT-treated spores requiring lysozyme for germination and colony formation originated from only a small portion of the non-UHT-treated spore population. This raised a question of whether the requirement for lysozyme was natural to the spores or was induced by the UHT treatments. However, these spores did not require lysozyme for germination before UHT treatment, which confirmed that the requirement for lysozyme had been induced by the UHT treatment. Only 1 to 2% of the spores were naturally sensitive to lysozyme; therefore, the mere addition of lysozyme to the plating medium did not permit the enumeration of all survivors. Treatment of UHT-treated spores with ethylenediaminetetraacetate (EDTA) sensitized the spores to lysozyme and increased by 10- to 100-fold the number of survivors that were detected on a medium containing lysozyme. Under the heating conditions used, spores that were naturally sensitive to lysozyme and spores that required EDTA treatment were equally heat resistant. The recovery of severely heated Clostridium perfringens spores was greatly improved if the enumeration medium was supplemented with lysozyme (1,3,4). Lysozyme did not increase the colony counts of heat-activated spores, which suggested that the requirement for lysozyme was not natural to the spores but had been induced by the heat treatments (injury). For C. perfringens spores heated at ultrahigh temperatures (UHT), however, the spores responding to lysozyme in the recovery medium were derived from only a small portion (1 to 2%) of the non-UHT-treated spore population (1). Had these spores naturally required lysozyme for germination, a 1 to 2% difference between the colony counts of heat-activated spores enumerated on a medium with or without lysozyme would not have been detected by the agar plate count method (plating duplicate samples). It was not certain, therefore, that the spores had actually been injured by UHT treatment. When UHT-treated C. perfringens spores were enumerated on a medium containing lysozyme, the time-survivor curves were biphasicconcave (1). Such curves are typically observed for the inactivation of spore populations composed of two types of spores. The biphasic l Paper no. 4224 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C. time-survivor curves were observed only when lysozyme was present in the medium and when lysozyme germinates C. perfringens spores (3,4), which suggested that the two types of spores differed in their sensitivity to lysozyme or in the heat resistance of their outgrowth systems. The distinction is vital to the development of thermal processes capable of lowering to an acceptable level the number of viable C. perfringens spores in a food. Unless all injured survivors are sensitive to lysozyme, the mere addition of lysozyme to the recovery medium is inadequate for their detection. The findings presented here show that: (i) the requirement for lysozyme was induced by heating; (ii) within a spore population, the spores differed in their sensitivity to lysozyme; and (iii) enumeration of all survivors required that the heated spores be sensitized to lysozyme prior to enumeration on a medium containing lysozyme. MATERIALS AND METHODS Maintenance of test organisms, composition and preparation of media, preparation of spore suspensions, heat treatments, and methods for enumeration of surviving spores have been described (1). Germination of lysozyme-sensitive spores in a complex medium. An Germination of lysozyme-sensitive spores by lysozyme. Non-heat-activated strain 8798 spores were suspended in 50 mM sodium phosphate buffer (pH 7) with or without 18 U of lysozyme per ml. The spores suspended in each medium were incubated at 45 C for 1 h and then heated at 75 C for 20 min to kill any germinated spores and heat activate the ungerminated spores. The spores were then washed twice, suspended in distilled water, heated at 105 C, and enumerated on TYCS plus lysozyme (18 U/ml). EDTA sensitization of UHT-treated spores. Duplicate capillary tubes, each containing 0.05 ml of UHT-treated spore suspension, were crushed in 100 ml of 10 mM sodium ethylenediaminetetraacetate (EDTA; pH 9.5) and held at 45 C for 1 h (2). The spores then were diluted in 0.1% peptone water (plus 0.02% Antifoam B, Sigma Chemical Co. St. Louis, Mo.) and enumerated on TYCS with or without lysozyme (18 U/ml). RESULTS AND DISCUSSION Injury during UHT treatment. We reported (1) that UHT-treated C. perfringens spores requiring lysozyme for colony formation appeared to derive from only 1 to 2% of the non-UHT-treated spore population. This portion of the total population was so small that it was not certain if the requirement for lysozyme was induced by UHT treatment (injury) or was natural to the spores. If these spores naturally required lysozyme for germination and colony formation, a medium containing lysozyme should be effective for the enumeration of all survivors. However, if these spores were injured during UHT treatment, the remaining spores also may have been injured but were not detected on TYCS plus lysozyme. The mere incorporation of lysozyme in the enumeration medium may not be sufficient for the detection of all survivors. To determine whether spores requiring lysozyme after UHT treatment were able to germinate normally before UHT treatment, heatactivated strain 8798 spores were incubated in TYB, heated to kill germinated spores, and then UHT treated. Spores incubated in water instead of TYB were treated similarly. Colony counts of non-UHT-treated spores incubated in water or TYB were 1.3 x 10'/ml and 7 x 107/ml, respectively, indicating that 94% of the spores had germinated in TYB (Fig. 1). If 1 to 2% of the spores had been unable to germinate normally in TYB (i.e., required lysozyme for germination), they should constitute 17 to 33% of the spores remaining after this extensive germination. This change in the composition of the spore population would be reflected in the time-survivor curves, because only the second phase of the curves represented a response to lysozyme (1). However, the time-survivor curves for spores held in TYB or water were identical, indicating that the relative composition of the spore suspension was unchanged by germination in TYB. The spores that required lysozyme after UHT treatment had been able to germinate normally before UHT treatment. This confirmed that the spores had been injured by the UHT treatments and suggested that other spores also may have been injured but were not detected on TYCS plus lysozyme. Basis for the biphasic time-survivor curves. The time-survivor curves were biphasicconcave only when lysozyme was used in the enumeration medium (1). Lysozyme reportedly germinated injured C. perfringens spores (3,4). If lysozyme germinated only some of the spores, the biphasic survivor curves must represent the rapid injury of spores not sensitive to lysozyme and the slower inactivation of the outgrowth system of spores sensitive to lysozyme. Alternatively, if all of the spores are sensitive to lysozyme, the two portions of the survivor curves reflect inactivation of the outgrowth systems in spores that differ in heat resistance. In the latter case, many of the heated spores germinated by lysozyme would not complete outgrowth, and the number of spores germinated by lysozyme should be up to 100-fold greater than the number of survivors enumerated on a medium containing lysozyme. To test this, strain 8798 spores were heat activated and then UHT treated at 105 C for 2.5 or 5 min. Survival and germination activity of non-UHT-treated and UHT-treated spores were determined. The data in Table 1 show that UHT treatment greatly reduced the number of spores able to germinate in TYB. Germination of UHT-treated spores in TYB plus lysozyme, however, was no greater than in TYB without lysozyme, and the extent of germination in either medium was similar to the level of survival measured on TYCS plus lysozyme. This indicated that the majority of the UHT-treated spores were not sensitive to lysozyme and suggested that the biphasic nature of the time-survivor curves was based on differences among the spores in their sensitivity to lysozyme rather than on differences in the heat resistance of their outgrowth systems. Results of an earlier study (1) suggested that a portion of strain 8798 spores was naturally sensitive to lysozyme. The spores required heat activation for normal germination, and germination of a small percentage of the spores by lysozyme was indicated by the higher colony counts of non-heat-activated spores on TYCS with lysozyme than on TYCS without lysozyme. The experiment was repeated with 10 replicates for each medium so that the results could be statistically analyzed. In the absence of lysozyme, 3.7% of the spores formed colonies; with lysozyme in the medium, 5.9% of the spores germinated and formed colonies. This increase of 2.2 percentage units was statistically significant of P = 0.025 (t test, degrees of freedom = 13) and was similar in magnitude to the percentage of heat-activated spores that had appeared to be unique in their sensitivity to lysozyme or the heat resistance of their outgrowth systems (1). In another experiment, unheated strain 8798 spores were incubated in a lysozyme-phosphate buffer mixture to allow the germination of any Clostridium perfringens NCTC 8798 spores to lysozyme on subsequent inactivation kinetics and recovery on TYCS plus lysozyme (18 U/ml). VOL. 27, 1974 lysozyme-sensitive spores prior to heat activation and UHT treatment. Spores held in phosphate buffer without lysozyme served as a control. The lysozyme pretreatment did not affect colony counts of heat activated-non-UHT-treated spores (data not presented) or the inactivation kinetics of spores not responding to lysozyme in the plating medium (represented by the first phase of the survivor curves, Fig. 2). However, for lysozyme-pretreated spores, the first phase of the survivor curve extended to a lower level of survivors before the response to lysozyme in TYCS (indicated by the second phase of the time-survivor curve) was observed. Extrapolation of the second phase of each survivor curve indicated that 97% of the spores that normally would have been germinated by lysozyme in TYCS already had been germinated by lysozyme during pretreatment. This confirmed that a small portion of the unheated spores was naturally sensitive to lysozyme, and suggested that it was recovery of the surviving fraction of these spores that was enhanced by the use of lysozyme in TYCS. Generally, bacterial spores are resistant to lysozyme and require some sensitization treatment (5). Bacillus megaterium ATCC 9885 spores appear to be the only exception (6). However, natural sensitivity to lysozyme by a very small portion of a spore population, as shown here for C. perfringens NCTC 8798 spores, would be easily overlooked. The natural sensitivity of B. megaterium ATCC 9885 spores may not be as unique as the large percentage of the spores having this trait. To confirm that the biphasic time-survivor perfringens spores were sensitized lysozyme by EDTA (2). The EDTA treatment did not influence the recovery of spores on TYCS lacking lysozyme, but increased by up to 100-fold the number of survivors detected on TYCS plus lysozyme (Fig. 3). This was observed for strain 8798 spores heated at 105 or 120 C (Fig. 3) and for two other strains of C. perfringens spores ( Table 2). The time-survivor curves for EDTAtreated spores enumerated on TYCS plus lysozyme were linear and unbroken, which indicated that all of the spores had been sensitized to lysozyme and that the biphasic nature of the time-survivor curves resulted from the resistance of many of the survivors to lysozyme. When survivors were enumerated on TYCS plus lysozyme, the D values (decimal reduction times: times required for a 90% decrease in the number of viable spores) for spores requiring EDTA treatment were similar to those for spores naturally sensitive to lysozyme. This indicated that, for the particular heating conditions used, the two types of spores were equally heat resistant. The results demonstrate that many of the C. perfringens spores surviving UHT treatment were injured and required lysozyme for germination and colony formation, but the majority were not sensitive to lysozyme; maximal recovery of survivors required that the heated spores be sensitized to lysozyme prior to enumeration. In the absence of such a treatment, more than 90% of the survivors were not detected, and the actual heat resistance of these spores was unknown. The usefulness of lysozyme for the recovery of injured spores may not be limited to C. perfringens spores. Alderton, Chen, and Ito (Abstr. Annu. Meet. Amer. Soc. Microbiol., p. 17, 1973) reported that lysozyme increased the recovery of heated C. botulinum spores. As with C. per-fringens spores, however, the time-survivor curves were linear and unbroken when survivors were enumerated on a medium lacking lysozyme, but were biphasic-concave when lysozyme was used in the medium. This suggests that the effective use of lysozyme for the enumeration of injured C. botulinum spores, or spores of other species, may require that the spores be sensitized to lysozyme before the survivors are enumerated.

v3-fos

2020-12-10T09:06:03.885Z

1974-12-01T00:00:00.000Z

237230452

s2

Factors Affecting the Sensitivity of Limulus Lysate Limulus lysate clots when mixed with picogram quantities of endotoxins. The sensitivity of the lysate was improved 100-fold by the removal of an inhibitor and addition of divalent cations. The methods developed in this investigation eliminated much of the seasonable variability of the lysate, improved the heat stability after lyophilization, and made it possible to use the lysate with saline solutions. Limulus lysate clots when mixed with picogram quantities of endotoxins. The sensitivity of the lysate was improved 100-fold by the removal of an inhibitor and addition of divalent cations. The methods developed in this investigation eliminated much of the seasonable variability of the lysate, improved the heat stability after lyophilization, and made it possible to use the lysate with saline solutions. Limulus lysate (an aqueous extract of the amebocytes obtained from the blood of the horseshoe crab, Limulus polyphemus) clots when mixed with picogram quantities of endotoxins which are lipopolysaccharide components of the outer cell-wall layer of gram-negative bacteria. Since the initial description of the lysate method for the detection of endotoxins (7), it has become apparent that this biologically active material may serve as a useful tool in several scientific disciplines. It has already been used for the detection of endotoxemia (6,8) and bacteriuria (4), and for the diagnosis of gram-negative spinal meningitis (10). The use of the Limulus test for the detection of endotoxins in radiopharmaceuticals and biologicals has already been demonstrated (2) and in the future it may be routinely used by pharmaceutical companies for this purpose. It is foreseeable that this test can be used to determine pollution in natural waters and to detect gram-negative bacterial contamination in food products. Lysate has been used in our laboratory to measure the horizontal and vertical distribution of bacteria in oceanic waters. As a laboratory tool, Limulus lysate may be useful in studying why endotoxins are so biologically active and for obtaining a better understanding of the biochemical events which occur in primitive clotting systems. Before the full potential of Limulus lysate can be realized, the biochemical mechanism involved in the lysate-endotoxin reaction must be more fully understood and the factors affecting this reaction must be detailed. Young et al. (16) have shown the reaction to be enzymatic consisting of endotoxin activation of a high-molecular-weight enzyme followed by its conversion of a low-molecular-weight clottable protein to a I Contribution no. 3308 from the Woods Hole Oceanographic Institution, Woods Hole, Mass. 02543. gel. Solum (14,15) has purified and characterized the clottable protein and gel protein. One of the serious objections to the use of the Limulus lysate method for the detection of endotoxins stemmed from the fact that the biological activity of the lysate varied from batch to batch and the factors controlling this variability were not understood. This report provides an explanation for the variable potency of lysate and corrective methods for improvement. MATERIALS AND METHODS Preparation of Limulus lysate. Horseshoe crabs were obtained from the Woods Hole Marine Biological Laboratory. Crabs were bled three to five times per week with an average of 50 crabs per bleed. Lysate was prepared by the method of Jorgensen and Smith (5). Assay of lysate. To test the sensitivity of the lysate, 0.1 ml of lysate was mixed with 0.1 ml of endotoxin in a pyrogen-free disposable test tube (10 by 75 mm) (Becton-Dickinson, Rutherford, N.J.). This mixture was incubated at 37 C for 1 h and scored as positive if the lysate formed a firm clot which would not break when the tube was slowly inverted 180 degrees. Endotoxins. A Klebsiella pneumoniae standard endotoxin preparation was furnished by the Food and Drug Administration and contained 100 ng of endotoxin per ml when reconstituted with 10 ml of pyrogen-free distilled water (Travenol Laboratories, Deerfield, Ill.). From this stock solution, which is stored at 5 C, a 1-ng/ml solution was prepared freshly each day with pyrogen-free water and from which a dilution series was made. Removal of inhibitor from lysate. Equal volumes of lysate and organic solvents were shaken for 1 h on a rotary shaker at 5 C. After shaking, the mixture was centrifuged (4,000 x g) and the aqueous phase was withdrawn and titered with various concentrations of endotoxin. All solvents used were reagent grade. Lyophilization and storage of the lysate. Before lyophilizing, 5 ml of lysate was dispensed into 10-ml serum vials equipped with a split rubber stopper. The lysate was then frozen at -74 C, lyophilized, and sealed under 10-gm vacuum in a Virtis 10-800 freezedryer (Gardiner, N.Y.) equipped with a stoppering plate. Usually 99 vials of lysate were dried at a time with a drying time of approximately 36 h. The moisture content of dried lysate was 2%. After drying the vials were banded with metal seals and stored at -74 C. Polyacrylamide gel electrophoresis. To demonstrate the nature of the inhibitor, samples of lysate before and after chloroform extraction were subjected to polyacrylamide disc gel electrophoresis at pH 9.5 according to Ornstein (11) and Davis (3). Gels were fixed in 12.5% trichloroacetic acid and stained with a 1:20 dilution in 12.5% trichloroacetic acid of a 1% solution of Coomassie blue R (Sigma Chemical Co., St. Louis, Mo.) according to Chrambach et al. (1). A duplicate gel was stained for lipoprotein by immersing it in a saturated solution of Sudan black B (Matheson, Coleman, and Bell, East Rutherford, N.J.) in ethylene glycol (13). Gels were destained in the first case with 12.5% trichloroacetic acid and in the latter with ethylene glycol. RESULTS Using procedures for lysate preparation similar to those described by Jorgensen and Smith (5) and Reinhold and Fine (12) the sensitivity of the lysate prepared in this laboratory varied significantly throughout the year. When these methods were used, 90% of the lysate prepared in our laboratory in the summer of 1972 formed a firm gel with 0.1 to 1.0 ng of endotoxin per ml. Yet in the summer of 1973 and 1974 very little of the lysate prepared would clot with this range of endotoxin. Lysate prepared during the winter of 1973 would not even gel with 100 ng of endotoxin per ml. The sensitivity of the lysate was improved and much of the variability eliminated by removing an inhibitor. This inhibitor was removed with a variety of water-immiscible organic solvents. Listed in their order of effectiveness they are as follows: chloroform > ethylene chloride > methylene chloride > ethyl ether > carbon tetrachloride > trichloroethylene > toluene > hexane. Solvents which did not effectively remove the inhibitor include nbutanol, isoamyl alcohol, phenethyl alcohol, amyl acetate, methyl isobutyl ketone, dichlorobenzene, and benzaldehyde. All solvents were shown to be free of endotoxin. Since chloroform was most effective it was routinely used in these experiments. Even after chloroform extraction the lysate did not achieve maximal sensitivity until a divalent cation was added. Addition of 0.02 M Ca2+, Mg2+ or Mn2+ were equally effective in increasing the sensitivity of the lysate. NaCl (0.154 M) was also added to decrease the turbidity of the blank. The effect of chloroform extraction and addition of divalent cations to the lysate is summarized in Table 1. Chloroform extraction and addition of divalent cations did not completely eliminate the seasonal variability of the lysate. The lysate prepared during the summer months was more sensitive than that prepared during the winter. In the summers of 1973 and 1974 the lysate clotted with 0.02 to 0.08 ng of endotoxin per ml while the lysate prepared during the winter of 1973-1974 required 0.1 to 0.4 ng of endotoxin per ml for clot formation. Chloroform extraction altered the stability of the lysate. Before chloroform extraction lysate was stored for periods up to 12 months at -74 C without loss of activity. Chloroform-extracted lysate occasionally but not always decreased in activity after 3 to 6 months of storage at -74 C. Nonchloroform-extracted lysate was stable at 5 C for more than a week. Chloroform-extracted lysate was unstable at room temperature and frequently lost activity when stored at 5 C overnight. To avoid loss of activity the chloroform-extracted lysate was routinely kept on ice during the working day. Chloroform-extracted lysate when lyophilized was more stable than nonchloroform-extracted lysate. Lyophilized lysate has been stored in this laboratory for a period of more than 12 months without loss of activity. No loss of activity of lyophilized lysate was found even when stored at 37 C for 2 months. Dried lysate has been shipped without refrigeration from this laboratory to other laboratories in both Europe and United States and investigators receiving the dried lysate reported that it had not deteriorated during shipping. When chloroform-extracted lysate was used with solutions containing 0.154 M NaCl or greater, the sensitivity of the lysate was frequently decreased. When 0.02 M Ca'+ or other divalent cations were added to the chloroformextracted lysate the sodium inhibition was eliminated. The fate of the inhibitor during chloroform treatment is not completely understood. When lysate was treated with chloroform, a large precipitate formed particularly at the interface. It appears that the inhibitor was precipitated and/or denatured by solvent treatment. When the residue from evaporation of the chloroform phase was resuspended in 0.154 M NaCl and tested with treated lysate, no inhibition was found. Comparison of gels stained with Coomassie blue failed to show a difference in protein pattern between chloroform and nonchloroform-treated lysate. A distinction could be seen if gels were stained with Sudan black B. Nontreated lysate showed a single band stainable with Sudan black (mobility = 0.13 relative to bromphenol blue, the tracking dye); the chloroform-treated lysate lacked such a band. Since this compound was water soluble and stained with Sudan black B, it is likely a lipoprotein. DISCUSSION In our laboratory it was found that high quality lysate could not be routinely prepared from horseshoe crabs collected on Cape Cod on a year-round basis using methods previously described in the literature. The use of the supplemental procedures which we used may not be necessary if the horseshoe crabs are collected during the summer months from other geographic localities. In our laboratory chloroform extraction and the addition of Ca2+ improved the sensitivity of the lysate more than 100-fold and has made it possible to prepare lysate on a year-round basis. All evidence indicates that chloroform extraction removes an inhibitor but the role of such an inhibitor is not clear. Higher concentrations of inhibitor may be present in the blood of the crab in the winter than in the summer months but this has not been experimentally verified. Possibly the concentration of the inhibitor remains relatively constant while the concentration of the clotting enzyme and/or the clottable protein is decreased during various, times of the year. If this is the case then even small amounts of inhibitor might prevent clotting of the lysate during periods when the concentration of active proteins is low. The in vivo function of the inhibitor in Limulus blood is not known. Quite possibly it acts at one or more steps involved in the clotting reaction which have been detailed by Young et al. (16) and include enzyme activation by endotoxin followed by enzymatic conversion of clottable protein to a gel. The inhibitor could limit the activation of clotting enzyme by tying up endotoxin therefore reducing the lysate's sensitivity. Levin et al. (9) have shown serum of human blood to contain a protein which binds endotoxin reversibly thus interfering with detection of endotoxin either added to or already present in human blood. Endotoxin could be detected with Limulus lysate when human serum or plasma was extracted with chloroform (9). The inhibitor in human blood is chloroform sensitive as is the inhibitor in Limulus blood and both may function as endotoxin-binding substances. Another possibility is that the inhibitor in Limulus blood interferes with the action of endotoxin-activated enzyme on clottable protein. Solum (15) has reported that trypsin in the absence of endotoxin gelled lysate or purified clottable protein. Trypsin inhibitor will block the trypsin-catalyzed reaction but not the endotoxin-dependent reaction (15). The clotting enzyme may be similar to trypsin in action and controlled by an inhibitor highly specific like trypsin inhibitor. Interference with the polymerization of gel protein into a gel may be another possible function of the inhibitor. That more than one inhibitor, each chloroform sensitive, is involved cannot be ruled out. Preliminary studies on the nature of the inhibitor in Limulus blood suggest that it is a lipoprotein. Polyacrylamide disc gel electrophoresis of lysate before and after chloroform extraction showed the disappearance of a protein band stainable with Sudan black, a lipoprotein stain, after solvent treatment. Additional experiments are in progress to confirm the lipoprotein nature of the inhibitor. The addition of divalent cations to lysate increases its sensitivity following solvent treatment. CaCl2 (0.02 M) or MgCl, (0.02 M) appear equally effective in lysate improvement. The role divalent cations play in the lysateendotoxin reaction is not known. Calcium could function at the enzyme activation step in conjunction with endotoxin, the reaction of activated enzyme with clottable protein, or the polymerization step resulting in a gel. Further studies are needed to determine the site(s) of action for divalent cations in the clotting reaction.

v3-fos

2018-04-03T00:40:21.672Z

1974-09-01T00:00:00.000Z

13643118

s2

Improved isolation and differentiation of enterococci in cheese. Further documentation of an enterococcus selective differential (ESD) medium was obtained in isolations from eight different cheeses. An improved differentiation of tetrazolium salt (2, 3, 5-triphenyl tetrazolium hydrochloride [TTC])-reducing strains of Streptococcus faecalis from TTC-nonreducing or TTC-faintly-reducing Streptococcus faecium was attained. The sensitivity of the medium was evaluated in comparison with that of KF streptococcal, Pfizer selective enterococcus (PSE), the medium of Reinbold, Swern, and Hussong (RSH), and the medium of Saraswat, Clark, and Reinbold (SCR). Selective counts, rate of colony formation, and ease of isolation and differentiation of colonies were examined. The specificity of the medium was also investigated. ESD supported the fastest rate of growth and the maximum size of colonies; counts in this medium were in most cases possible with 17 h of incubation, whereas the other media required 24 to 48 h. A presumptive identification of 1,077 isolates by four biochemical tests disclosed that SCR, RSH, and ESD selected high, comparable percentages of strains that approximated most closely the typical description of enterococci (66, 60.1, and 58%, respectively). Low percentages (21.1 and 30.7%) were yielded by KF and PSE. The utility of ESD for a rapid, presumptive identification of enterococci was confirmed by serological and biochemical testing of color TTC-differentiated colonies isolated from 18 cheeses. A selective medium suitable for the isolation of enterococci was previously developed by incorporation of a low sodium azide concentration (0.01%) and a high alkaline (pH 9.6) into a manganese-deficient nutrient base (5). In the initial evaluation of this medium, known pure strains of bacteria were used. In the present study, we extended the utility of the medium by including in its composition a tetrazolium salt indicator effecting presumptive speciation of the enterococcal isolates. We also tested the selective and differential capacities of the medium on various cheeses. Comparative isolations of enterococci were conducted by using several standard media. We made observations on the sensitivity of enterococcal recovery; selective counts, rate of colony formation, ease of isolation, and differentiation were considered. For an evaluation of specificity of the medium, we carried out a comparatively presumptive identification of cheese isolates. Finally, we examined the practical features of the medium in a few parallel applications of its solid and liquid form. MATERIALS AND METHODS Culture media. The medium tested in this study, designated enterococcus selective differential (ESD), was that which was described in a previous study (5), modified to include a tetrazolium salt indicator. After addition of sterile agar, the temperature of the medium was decreased to 50 C, and 5 ml of a 2% 2,3,5-triphenyl tetrazolium hydrochloride (TTC), sterilized previously in flowing steam for 30 min, was added. This medium was also tested as ESD broth (agar omitted). In this case, the sugar was autoclaved separately as a concentrated (10%) solution and its final concentration was reduced to 1% (the glucose level of Barnes TG (1) medium with which ESD was compared). The following agar media were used in addition to ESD: KF-streptococcal (BBL), Pfizer selective enterococcus (PSE), the medium of Reinhold, Swern, and Hussong (RSH; 14), and the medium of Saraswat, Clark, and Reinbold (SCR;15). Standard methods agar (SMA;BBL) was used as a nonselective control. Yeast extract (YE) agar was used as recovery and maintenance medium for all isolates from cheese. The composition of this medium was: yeast extract (20 g), K2HPO4 (2 g), MgSO4.7H2O (0.1 g), glucose (2 g), agar (15 g), and distilled water to 1,000 ml. The final pH was adjusted to 6.8. The same composition, but without agar, was used as YE broth for preparation of inocula in the presumptive identification of cheese isolates. TTC reduction in ESD media. The kinetics of TTC reduction by enteroccocci were studied comparatively in the standard TG medium of Barnes (1) and ESD broth. For this purpose, the same concentra-inoculated with 3 drops of a 24-h culture. The reducing activity of known strains of enterococci, representing different physiological types, was examined visually every hour between 0 and 8 h, and at 24 h. Reduction was demonstrated by the appearance of magenta-colored triphenyl tetrazolium formazan. The reduced TTC was extracted with n-butanol. The optical density of the extracts was measured at 575 nm. Several concentrations of TTC were added to the selective ESD broth and were tested for a determination of the optimal concentration, i.e., the amount that was nontoxic and produced the best visible results. The same TTC concentrations were also incorporated in ESD agar plates which were then surface inoculated with the test organisms. At 0.01%, added to both liquid and solid media, no inhibition of cultures was noted, and the color of positive reactions was intense. Afterwards, this concentration was used routinely with both forms of ESD. Cheese samples analyzed. Eight different samples of cheese were used for the comparative isolation, enumeration, and presumptive identification of enterococci. An additional collection of 18 cheeses was used for the isolation, presumptive, and confirmed identification of isolates from the ESD medium only. All cheeses were obtained in retail packages from the local New York market. They represented cheese varieties differing as to country of origin, manufacturing procedures, species of animal that produced the milk manufactured into cheese, and environmental conditions prevailing at the time of manufacture and early ripening. All samples had been ripened for at least 60 days; some samples had been ripened for more than 6 months before they were marketed. Isolation and enumeration of enterococci. The handling, preparation, and analysis of the cheese samples were performed according to recommended methods (17). Serial, 10-fold dilutions were plated in SMA to determine the total viable count and in the selective media for comparative counting and isolation of enterococci. In the selective agar media, the size of inoculum was 0.1 ml; it was spread evenly on the surface of the agar using a bent glass rod. All cheese dilutions were inoculated in triplicate. The plates were incubated aerobically at 37 C. Counts were determined at 17, 24, and 48 h. Individual colonies were isolated by using the random sampling method of Harrison (9) after 48 h of incubation, and 30 to 60 colonies were picked per sample and medium. The counts were compared by the F test. For an assessment of sensitivity, the average size of 10 representative colonies, developed on the surface of each medium, was determined. The size or diameter of each colony was measured in 0.1-mm units on a Quanti Plate viewer (Kallestad Lab., Minneapolis, Minn.) apparatus utilizing dark-field lighting and a magnifying optical comparator, ordinarily used to measure immunoprecipitation patterns in agar gels. The relative sensitivity of the media was established by statistical analysis of the mean diameter of the enterococcus colonies by the Student-Newman-Keuls test. This is a test of multiple comparisons among means based on equal sample sizes (in this study, 10 colonies per plate), and it is derived from the principles of analysis of variance (16). Presumptive identification of enterococci. The cheese isolates obtained from the five selective media were inoculated in YE agar stabs. When growth was obtained, the cultures were placed in a refrigerator, where they were kept until further testing. For a presumptive identification of enterococci, the following five tests were performed: catalase activity, ability to grow on bile esculin medium (BEM), SF (Difco) medium, 0.1% methylene blue milk (MBM), and 6.5% NaCl broth. The latter four tests were performed and interpreted as a battery of tests. They were carried out according to the methods of Facklam and Moody (7). Catalase activity was tested in YE broth cultures. Inocula for all five tests were also grown in YE broth. The tests were adapted to utilize the multipoint inoculation system of Lighthart (12) and were read after 24 h of incubation at 37 C. The evaluation of relative specificity of the five media for enterococci was based on a statistical analysis (chi-square method) of the comparative identification data. Confirmed identification of enterococci. Colonies from 18 different samples of cheese, upon isolation from ESD agar, were transferred into serological tubes containing 2.5 ml of ESD broth. These cultures were incubated for 4 h at 37 C and then were examined for TTC reduction. The identification tests included the following. The morphology of isolates was examined by the gram stain. Presumptive verification was according to Sherman's criteria, the catalase reaction, and appearance on BEM (Difco). A screening for group D antigen was carried out by the Lancefield precipitation method in capillary tubes; for this test, a grouping antiserum was prepared in rabbits using the strain Lancefield D76 (S. faecalis var. zymogenes ATCC12958). Antigenic extracts for precipitation were produced from cells cultured in glucose Lemco broth according to Medrek and Barnes (13). Differentiation into species was determined in representative TTC-reducing and TTC-nonreducing strains, by testing for tolerance to potassium tellurite, fermentation of sorbitol, mannitol, arabinose, melibiose and raffinose, determination of folic acid requirement, substrate utilization of pyruvate, malate, and serine, and ability to grow in broth at 50 C. These tests were essentially performed according to Deibel (2,3). RESULTS TIC reduction in ESD. A summary of results is presented in Table 1. In the TG medium, the differentiation of typical S. faecalis from S. faecium by TTC reduction required 24 h of incubation. Intermediate enterococci, such as the epiphytic strains T-15, FMA 2, and FMA-11 (5), developed a positive reaction. These atypical S. faecium strains were confused with some of the, slowly reducing strains of S. faecalis. The reduction time of all known S. faecalis strains in ESD broth was less than 4 h. The optical density values, obtained from formazan extracts of cultures at 4 h, ranged approximately between 0.35 (strong TTC reducers, e.g., strain ATCC11700) and 0.12 (moderate Some of the colonies that developed on PSE failed to show the typical black halo of enterococci. The percentage of such atypical colonies varied from cheese to cheese; the lowest proportion that could be determined was 0% in the Kefalotyri sample and highest (51%) in the blue cheese sample. The recognition and enumeration of these atypical colonies on PSE, quite easy early in the incubation, became progressively difficult after 21 h as the black salts of hydrolyzed esculin surrounding typical colonies diffused through the agar. A meaningful comparison of colony sizes on all five selective media became possible after 48 h of incubation, when measurements could be made on KF, RSH, and SCR media. Table 2 indicates the average diameters of colonies that developed on each medium. Statistical analysis of the means (Student-Newman-Keuls test) disclosed that the colonies grown on KF, PSE, RSH, and SCR media did not differ in size significantly. However, the size differences between colonies grown on ESD and all other media were significant at the 95% level. Specificity of ESD for enterococci. A presumptive identification was carried out on 1,077 isolates. This total represented strains from all selective media and most of the cheese samples included ( Table 2). Thirteen strains (1.2%) showed a positive catalase test. Table 3 gives the distribution of strains that reacted positively in 4, 3, 2, 1, or none of the presumptive enterococcus tests. The results indicate that SCR, RSH, and ESD media selected the highest percentages of strains that reacted positively in all four presumptive tests (66, 60.1, and 58% respectively). In contrast, KF and PSE yielded low percentages (21.1 and 30.7, respectively). Substantially similar comparisions were made when the statistical evaluation was based on the results of only two of the four tests performed, i.e., reaction on BEM and tolerance to 6.5% NaCl; the combination of the 2 tests was found recently to be a reliable criterion for a presumptive identification of group D streptococci (6,8). A total of 936 other isolates from 18 cheese samples were obtained on ESD agar (Table 4). These strains were subjected to verification of identity. They were all gram-positive cocci and catalase negative; with some minor variations, they conformed to the Sherman's criteria. They could grow in the presence of bile salts and hydrolyzed esculin, and they demonstrated the group D antigen. Strains that reduced TTC on the ESD agar plates and showed TTC reduction in less than 4 h in ESD broth were confirmed as S. faecalis by the following pattern of biochemical characteristics: they could grow in the presence of 0.05% potassium tellurite; they could produce acid from sorbitol and mannitol, whereas they failed to produce acid from arabinose, melibiose, and raffinose; they grew well in folic acid assay medium without the addition of the cofactor; they utilized pyruvate, malate, and serine as energy sources; and they were unable to grow at 50 C. Strains that did not reduce TTC were confirmed at S. faecium by their intolerance to potassium tellurite, their production of acid from mannitol, arabinose and, with some exceptions, from melibiose. These strains required folic acid for growth. They could not utilize pyruvate, malate, and serine, and most of them were able to grow at 50 C. A few TTC-nonreducing strains showed these reactions but failed to ferment mannitol and arabinose. They were considered S. faecium var. durans (3). The enterococci of 8 of the 18 cheeses (Table 4) were also enumerated by using the membrane filter technique and ESD broth. The counts by this method were slightly higher than those obtained in the surface-inoculated ESD agar plates. This was. probably due to some cell loss that occurred during the spreading of inocula on the surface of the agar by means of glass rods. DISCUSSION S. faecalis shows strong TTC reduction, whereas S. faecium reduces TTC weakly (1). However, this presumptive differentiation can be equivocal due to variation in the intensity of TTC reduction among biotypes of S. faecalis and the occasional faint reactivity of S. faecium variants. Langston et al. (11), using Barnes' TG medium and method, were not able to distinguish atypical S. faecium strains, reducing TTC faintly, from S. faecalis. After 8 h of incubation the appearance of both types of cultures was similar. The same authors noticed that some differentiation could be made between the two biotypes of enterococci when observations of TTC reduction were made earlier than 8 h. In our study, the kinetics of reduction of ESD broth also provided a better criterion for differentiation than the mere ability to reduce TTC. The distinction between the two species (including atypical varieties of S. faecium) was attained in less than 4 h ( Table 1). Differentiation by ESD proved useful in the isolation and identification of enterococci from various cheeses (Tables 2 and 4). Of the five selective media used, ESD sup-ported the fastest rate of growth and the maximum size of colonies. Differential counts on this medium could be determined in 25 out of 26 cheeses analyzed as early as 17 h, whereas the relatively large size of the colonies made their enumeration and isolation convenient. Quantitatively, the selective counts on ESD at 48 h appeared roughly comparable to those on RSH and SCR, media well suited for isolating and enumerating enterococci in milk, cheese, and other dairy products (10,14,15). The previous observation that ESD allows good growth of a wide range of physiological types of enterococci (5) was coupled in this study with demonstration of a high degree of selectivity for this group of bacteria ( Table 3). Two of the selective media used, KF and PSE, showed too broad specificity, since they allowed the selection of large percentages of strains with a combination of characteristics atypical for enterococci. Both PSE and KF were found to lack in specificity towards enterococci previously (6,7). The present data indicate that the selectivity of KF and PSE is downgraded appreciably when enterococci are isolated from habitats such as cheese containing preponderant numbers of lactobacilli and related bacteria. In contrast, the suitability of ESD for such isola- a Symbols:+, More than 80% of colonies reduced TTC; -, more than 80% of colonies did not reduce TTC; ±, both reducing and nonreducing types were present in substantial numbers. tions was demonstrated in this study; the specificity of ESD for enterococci was shown to be equivalent to that of RSH and SCR media (Table 3). In comparison with other available media, the preparation of ESD agar presents the average laboratory with some inconvenience. Several component solutions are made up and sterilized separately. Their mixing requires aseptic conditions. The addition of hot agar solution and the lability of TTC demand attention and control of temperature. For adequate exclusion of cations, it is preferable to use deionized water and purified agar. Surface inoculation is more time consuming than preparation of pour plates. Most of these limitations, however, can be eliminated with the liquid version of the medium. Used in conjunction with the membrane filter technique, ESD broth presents the same convenience as other standard media utilized in this manner.

v3-fos

2020-12-10T09:04:12.385Z

1974-03-01T00:00:00.000Z

237233689

s2

Production of Flavine-Adenine Dinucleotide from Riboflavine by a Mutant of Sarcina lutea A study was made to develop a new method for the production of flavine-adenine dinucleotide (FAD) from riboflavine and adenine by a mutant of Sarcina lutea deficient in the enzyme adenosine deaminase. It was found that this strain could convert exogenously supplemented riboflavine to extracellular FAD. The yields of FAD were increased by addition of D-cycloserine in the culture medium. The culture conditions for FAD production were investigated under the addition of D-cycloserine, and increased production of FAD was observed with the addition of an appropriate amount of thiamine, acetate, and sodium ion. The yield of 0.7 g/liter was obtained in the optimal culture in 5 days. Accumulated FAD was readily isolated by adsorption chromatography and ion-exchange chromatography in a 70% yield. The use of flavine-adenine dinucleotide (FAD) as a biochemical and nutritional agent has been recently increasing, and the chemical, biochemical, and fermentative methods have been reported for its production. In the past, FAD was produced by extraction from the mycelium of Eremothecium ashbyii (10) or by the chemical synthesis from flavine mononucleotide (FMN) and adenosine monophosphate (AMP) (2). We previously showed that a large amount of FAD accumulated in culture fluid when a strain of Sarcina lutea was cultured in the medium supplemented with FMN, a wellknown precursor of FAD, and adenine (7). During the course of these studies, it was found that riboflavine was more favorable than FMN as the precursor for FAD production because of its low cost and easy separation from FAD. This paper deals with the fermentative method for FAD production from riboflavine and adenine. MATERIALS AND METHODS Organism. A purine-requiring and adenosine deaminase-less mutant ATCC 21881, which was derived from Sarcina lutea IFO 1099, was used for the fermentative experiments. Fermentation experiments. Unless otherwise noted, fermentations for FAD production were carried out as follows. The compositions of seed medium and fermentation medium are shown in Table 1. Seed media were distributed in 30-ml amounts to 500-ml flasks, sterilized, and inoculated with one loopful of cells of the S. lutea mutants. Cultures were incubated at 30 C. After 24 h of incubation, 0.5 ml of the seed medium was combined with 20 ml of the respective fermentation medium in a 500-ml shaking flask. All cultures were incubated at 30 C with reciprocal shaking (120 rpm, 8-cm stroke). After 24 h of incubation, a 2.5-ml solution of 0.5% adenine and 0.5% riboflavine was added, and incubation was continued for an additional 2 to 4 days. Culture broth was heated at 80 C for 3 min and centrifuged. The supernatant was employed in the determination of products. Methods of analysis. The assay of FAD and FMN was carried out by the manometric method (7) and the fluorometric method (11). Determination of growth was carried out as follows. The culture broth was diluted 80-fold the original volume with water, and the optical density at 660 nm was measured with a photometer (Hitachi EPO-B type). An absorbance of 1.00 represented 1.6 mg of dry cells per ml. Sucrose Kulka (3). easily isolated without separating FMN and ured by the method of Hirata and FAD since only traces of FMN were formed nine, hypoxanthine, AMP, and from riboflavine. Accordingly, it was predicted determined by measuring their that riboflavine might be a more favorable 260 nm after extracting their spots precursor for FAD production. tograms with 0.01 N HCl. Paper Screening of detergents. It was shown that is carried out on Toyo filter paper some detergents vent system containing isobutyric some amino stimulated fermentative pro-,al water (5:3, vol/vol). duction of amino acids (8) and nucleic acidwas purchased from Boehringer related compounds (6). The benefit of adding heim, GFR), adenine was pur-detergents to the medium is the increase in cell Co., Ltd. (Tokyo), and ribofla-permeability. The results of Fig. 1 show that the 'okyo Tanabe Seiyaku Co., Ltd. rate-limiting step of FAD synthesis may be chemicals used were of the best riboflavine kinase (adenosine 5'-triphosphate previously demonstrated that formation would be stimulated and yields of active precursor for FAD pro-FAD would be substantially increased. Thus, ever, a large amount of FMN the effect of detergents on FAD overproduction ged in the final culture broth ersion a t h e ofinal FMNuinturot was investigated. Cationic surfactants, such as cetyltrimethylammonium bromide (CTAB) or are the chemical structures of cetylpyridinium chloride inhibited both growth milar to one another, but their and FAD production at 0.01% concentration ysical characteristics are quite ( Table 2). Growth inhibition could be reversed the separation of a large by lowering the concentration of the addition, from FAD in the culture broth but FAD production could not be increased. rder to avoid the disadvantage Almost all antibiotics, such as penicillin and e decided to use riboflavine, a streptomycin, showed the same result as ionic in the biosynthetic pathway of surfactants. Overproduction of FAD occurred I serine was specific for FAD oversynthesis. The fact that the addition of D-cycloserine inhibited growth showed that FAD overproduction was not due to an effect of D-cycloserine on growth, but was due to some other unknown mechanism. In media with D-cycloserine, the amount of FAD accumulated in the culture broth was twice that accumulated in medium without D-cycloserine. Effect of addition time of D-cycloserine on FAD production. The correlation between FAD production and addition time of D-cycloserine was studied in detail (Fig. 2). Growth inhibition was observed when D-cycloserine was added within 24 h after inoculation, but FAD production was effectively stimulated. On the other hand, when it was added later, FAD production was appreciably depressed in spite of no inhibition of growth. This result suggests that FAD production is increased under conditions causing growth inhibition. Accordingly, the addition time of D-cycloserine should be at the early stage of FAD fermentation. When D-cycloserine was supplied within 24 h after inoculation, growth obtained was less than half of the maximal growth. Effect of D-cycloserine concentration on FAD production. The effect of D-cycloserine concentration on FAD production is shown in Fig. 3. Experiments were made to determine an optimal concentration of D-cycloserine at zerotime addition. As seen in Fig. 3, addition of D-cycloserine at the concentration of 80 ug/ml gave the optimal yield of FAD, but growth was nearly 60% of the maximal growth without added D-cycloserine. S. lutea was markedly 0.6r Ag/ml strongly inhibited growth but that of 40 gg/ml permitted normal growth. Formation of FAD from FMN or riboflavine by non-growing cells. To examine how D-cycloserine stimulates the accumulation of FAD from riboflavine, activities of FAD formation in the cells grown on the basal medium with D-cycloserine and in the cell supernatant were compared with those grown without D-Cycloserine ( Table 3). The cells grown on the medium without D-cycloserine formed a small amount of FMN, but not FAD, from riboflavine, while they synthesized a relatively large in the cells grown on the medium supplemented with D-cycloserine, FAD was equally synthesized either from FMN or riboflavine in reasonable yields. The cell supernatant had no activities of FAD formation, regardless of the addition of D-cycloserine to medium. These results suggest that the stimulation of FAD formation from riboflavine may not be caused by the leakage of enzymes from the cells, but by the improvement of permeabilities of substrate or products. Effect of culture conditions on FAD production. The culture conditions for FAD production were investigated under the addition of D-cycloserine. (i) Effect of precursors. Various kinds of precursors were added to the culture medium at 24 h after inoculation. As shown in Table 4, it is apparent that this strain has a de novo pathway for FAD formation because a small amount of FAD was made without addition of precursor. Guanine, a well-known precursor of riboflavine, inhibited growth and was not effective for FAD production, whereas adenine and riboflavine stimulated FAD production without inhibiting growth. But separate addition of adenine and riboflavine did not stimulate FAD production compared with their simultaneous addition. Accordingly, it is essential for FAD overproduction that adenine and riboflavine are simultaneously added to culture medium. Growth inhibition by guanine was reversed by adding adenine, but the amount of FAD accumulated was about 120 ug/ml, which was the same amount as that accumulated by the single addition of adenine. (ii) Effect of aeration. The effect of aeration was studied by changing the volume of medium in the flasks. Maximal production of FAD was obtained with 25 ml of medium or less in a 500-ml flask, which is equivalent to an oxygen absorption rate greater than 2.95 mmol/min (Table 5). (iii) Effect of thiamine. Table 6 demonstrates the effect of thiamine concentration on FAD production. A low concentration of thiamine supported good growth but not FAD overproduction. There was also a similar tendency with regard to growth and FAD production at a high level of thiamine. The optimal concentration of thiamine was limited to a narrow range which was about 0.5 ,ug to 2.0 ,ug/ml. (iv) Effect of sodium ion. Sodium ion was also found to exert a considerable effect on FAD production. Experiments were carried out to examine the changes during fermentation with or without sodium ion (Fig. 4). When sodium ion was removed from basal medium, growth was enhanced, but FAD production was strongly inhibited. It was estimated that the inhibition of sugar assimilation caused by the removal of sodium ion repressed ATP formation. As the result, FAD production was inhibited. (v) Effect of acetate. We reported that acetate stimulated FAD formation from FMN (7). A similar result was obtained when using riboflavine (Fig. 5). Omission of acetate from the medium reduced the FAD yield by about 80%. When an optimal concentration of acetate was added, rapid assimilation of sugar and no for- x, pyruvate. mation of pyruvate was observed. The definite mechanism responsible for this phenomenon has not been established. Changes occurring during fermentation. An example of the chemical changes which occurred during the fermentation of FAD under optimal conditions is given in Fig. 6. After an initial lag period of approximately 12 h, the logarithmic phase proceeded for a long period, and rapid consumption of sucrose was accompanied by growth. As growth slowed down, FAD production started and reached a maximum of about 700 gg/ml at 5 days. The characteristic pH change during the growth phase was a feature of this fermentation process. The rise in pH suggested the start of logarithmic phase, and the pH rose as high as 8.5. Assimilation of acetate and formation of ammonium ion from peptone contributed to the pH rise. After growth ceased, the pH decreased to 6.5 -7.0 and remained constant during the latter part of fermentation. During fermentation, only traces of FMN formed in the medium. Accordingly, FAD in the fermentation broth was easily isolated by an ordinary procedure using Florisil and ion-exchange resins in a 70% yield. Infrared spectrum of the product was identical with that of authentic FAD, and no other fluorescence compound was detected by paper chromatography. DISCUSSION Many purine-pyrimidine-related substances are produced by fermentation methods. These include a number of nucleotides, nucleosides, and their analogues. Practical methods for the synthesis of nucleotide derivatives from the corresponding bases were recently reported (5,9). The synthesis of 5'-inosinic acid, ATP, nicotinamide adenine dinucleotide, coenzyme A, etc., are the examples of these methods. In a previous paper the production of FAD by a microorganism was reported; i.e., a large amount of FAD was produced by S. lutea from FMN and adenine in a medium containing sucrose and salts (7). But this previous method had some disadvantages. Among them are the high cost of FMN and the difficulty of separating FMN from FAD during the isolation procedure of FAD. To overcome these disadvantages, we attempted to use riboflavine instead of FMN as a precursor for FAD production. Riboflavine was an inferior precursor in comparison to FMN, because the ability to convert riboflavine into FMN was low in S. lutea. In general, fermentative production of nucleotides is markedly affected by the cellular permeability of the microorganism used. Therefore, studies were performed on the removal of the permeability barrier of S. lutea. The permeability barrier is removed by controlling the levels of trace nutrients or metals, or by the addition of the agents affecting cellular permeability such as surfactants, antibiotics, etc. As described above, FAD production from riboflavine was stimulated only by the addition of D-cycloserine. An example of using D-cycloserine was shown for fermentative production of 5'-inosinic acid by Nara et al. (6). They reported that the most important condition was to add the antibiotics at a very early stage of fermentation. A similar observation was made on FAD production. The addition of D-cycloserine at later than 24 h of incubation resulted in a marked reduction of FAD yields. It was estimated that the stimulation with the addition of D-cycloserine was caused by the improvement of the permeability barrier through the change of cell wall because D-cycloserine is known as a compound which affects the structure of microbial cell wall (4). This was confirmed by the experiments shown in Table 3. The change of cell permeability caused by D-cycloserine allowed for a more rapid conversion of riboflavine into FMN, the most immediate precursor of FAD. But it was desirable for production and isolation of FAD that the accumulation of FMN was not accelerated since accumulated FMN was easily converted to FAD by FAD pyrophosphorylase (ATP: FMN adenyltransferase; EC 2.7.7.2) in this strain. From a practical point of view, the present method has considerable advantages over any other microbial process previously reported (7,10). One of the advantages is that a high concentration of FAD is accumulated in culture fluid and another is that only traces of FMN are formed. These characteristics allow for the purification of product in a high yield without tedious procedure. Thus, the method presented here is considered to be a very advantageous one for FAD production.

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Conventional and Reduced Tillage Methods in Corn Production This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1974 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. D. Michael Powell, Agricultural Engineering Robert J. Raney, Research Agronomist Reduced tillage practices may provide benefits in corn production. Applying reduced tillage methods will result in reduced operating expenses and time spent in fields. By retaining surface cover, it will reduce soil erosion and may increase the percentage of rainfall stored as soil moisture, thus providing ready moisture for seed germination and early crop growth. However, there are potential hazards associated with reduced tillage. A weed problem may develop after several years of reduced tillage on a continual ba-;ls. Herbicide and/ or cultivation may not be able to control all weeds. Yields then may suffer, thereby reducing the economic incentives ofreduced tillage. This study reports a comparison of yields, weed control, and production cost for conventional andreduced tillage practices. The tests were conducted on Irrigated corn for 8 years (1974)(1975)(1976)(1977)(1978)(1979)(1980)(1981) at the same location on the Scandia Irrigation Experiment Field in North Central Kansas. This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. Materials and Methods Tests were made using four tillage systems (Table 1} in a split-plot design with four replications. Subplots were composed of cultivation when necessary and no cultivation. The D-P-D-S and D-D-S plots were 100 feet by 15 feet and planted with a 6-row planter. The D and N plots were 100 feet by 10 fee. and planted with a 4-row Buffalo Till planter. Herbicides were applied to all tillage treatments. Treatments D-P-D-S, D-D-S, and D were banded at planting with Lasso/ Atrazine (2.5 qts. and 2lbs/acre) in 20 gaUons of water per acre in 1974 and 1975. 2,4-D was broadcast before planting on theN plots at 1.5 pints and 1.0 pints per acre during 1974 and 1975, respectively. Lasso/ Atrazine, at the above rates, was broadcast to all treatments after planting with 20 gallons of water per acre for 1976 through 1978. Beginning in 1979 through 1981 the water was increased to 40 gallons per acre and broadcast after planting to all plots. The rates of Lasso/ Atrazine remained the same ex- { Fertilizer and insecticide, Furadan at 10 pounds per acre, were applied uniformly to all plots. Results and Discussion Corn hybrids and yields are reported in Table 2. Prior to 1979 the yields indicated significant differences among tillage methods with the no preplant tillage (N} being inferior. However, the 8-year summary indicates no significant differences among tillage methods. The trend of a significant difference prior to 1979, then no significant difference for the 8 -year (1981) summary Is reflected in the plant population (Table 3) . This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. In 1979, two changes were made that may account for this reversal of trend. Prior to 1979, there was a significant difference in plant population attributed to the difference in planters. Adjustments to the Buffalo Till planter in 1979 provided comparable plant populations In 1979through 1981. Also, 1979, herbicides were applied in 20 gallons of watei per acre, then beginning in 1979 the water was increased to 40 gallons per acre. The additional 20 gallons of water per acre may have provided better herbicide-soil contact thereby providing equivalent weed control among tillage methods from 1979 through 1981. (Table 4) . Either or both of these changes made in 1979 may be responsible for the reversal of the 6-year trend in yields and plant populations establishedin 1979. There were no significant differences among tillage methods for yields or plant populations for the 8-year period of this study. The practice or absence of cultivation was not significant between tillage methods Year 1974 1975 1976 1977 1978 1979 1980 1981 Avg.

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Studies on the cecal microflora of commercial broiler chickens. A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO(2) and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes. A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO2 and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes. There is little information available describing the predominant kinds of bacteria occurring in the intestinal tract of the commercial broiler. Studies on layer and broiler chickens have shown that the cecum contains the largest number of bacteria, most of which are strict anaerobes (2,4,18,22,23). Barnes and Impey (2,3) have isolated from poultry ceca (chickens, turkeys, pheasants, and ducks) several groups of anaerobic streptococci as well as gram-negative and gram-positive nonsporeforming anaerobes including species of Bacteroides, Fusobacterium, Eubacterium, Propionibacterium, and Bifidobacterium. In the few remaining studies concerned with chicken microflora, characterization of the cecal bacteria was limited to only a few groups which were cultured on selective media. In contrast, we previously observed that using nonselective rumen fluid media and strict anaerobic methods devised for rumen bacteria (20) allowed a large percentage of anaerobes to be isolated from the cecum of "laboratoryreared" broilers (5-week-old). Ninety percent of the microflora cultured in this prior study was composed of facultatively anaerobic cocci and streptococci and strictly anaerobic species of Peptostreptococcus, Propionibacterium, Eubacterium, Clostridium, Bacteroides, and unidentified gram-negative organisms. The work reported here extends our initial studies on the major groups of bacteria isolated from the chicken to include the cecal microflora of broilers which were reared under commercial husbandry conditions. Data on variability in microbial populations among samples and some growth requirements of the predominant anaerobes are also presented. MATERIALS AND METHODS Animals. Five-week-old cockerels (White Cornish cockerel x White Rock hen) maintained on as antibiotic-free grower ration (containing a coccidiostat) were obtained from a commercial broiler facility (Foster Poultry Frms, Livingston, Calif.). Cecal samples (3 to 4 g wet weight) were taken from animals (0.9 to 1.0 kg) which had been killed by CO2-asphyxiation. Culture methods and isolation of cecal bacteria. Direct microscopic counting, anaerobic culture techniques, sampling procedure, and nonselective anaerobic roll tube media used in this study were the same as 439 440 SALANITRO, BLAKE, AND MUIRHEAD previously described (20). Portions of 10-9 dilutions of the cecal samples were inoculated into roll tubes of M98-5 media. After 6 days of incubation at 37 C, 300 colonies were picked from roll tubes (50 to 60 colonies per tube) inoculated with cecal samples from six individual birds. The colonies were subcultured to slant media of similar composition (20). Mixed culture isolates were purified by restreaking on M98-5 media (15). Initial criteria used for grouping pure cultures of strains were morphology (phase contrast microscopy), Gram stain reaction, oxygen sensitivity (facultative anaerobes or strict anaerobes), growth bn glucose, and fermentation products formed from glucose. Media and methods used for these tests have been given (20). Carbohydrate fermentation, physiological tests, and identification of isolates. Tests to determine the ability of representative strains of anaerobes from bacterial groups in Table 1 to ferment several carbohydrates, to hydrolyze esculin and starch, to reduce nitrate, and to produce indole, H2, and H2S were performed. The basal media and methods used in these tests have also been described (20). Identification of strains was based on comparison of these various physiological features with those of known poultry isolates (2,3), classification schemes of Holdeman and Moore (15) and other published data on anaerobes. Analytical method for fermentation acids. Fermentation acids (formic, acetic, propionic, butyric, APPL. MICROBIOL. lactic, and succinic) elaborated in glucose-containing media were analyzed by a gas chromatographic procedure adapted from the method of Lambert and Moss (16) for the preparation of butyl esters. An estimate of the amount of each acid formed was determined from standard curves of authentic butyl esters (obtained from Pfaltz & Bauer, Inc.). The gas chromatograph used was a Hewlett-Packard model 5754B equipped with flame ionization detector an digital integrator. Ethanol in culture media was determined enzymatically with alcohol dehydrogenase (Sigma Kit no. 331-UV). Growth characteristics of the various strains were determined in media containing basal medium plus added components. The basal medium consisted of mineral solutions 1 and 2, glucose, Na2CO%, cysteinehydrochloride, and NaS at the same concentrations as given for IM. The following constituents were included to give the final concentrations (wt/vol, vol/vol, or mM): hemin (0.0002%), Trypticase (0.2%), yeast extract (0.2%), rumen fluid (CRF 2, 20%), volatile fatty acid mixture (29.5 mM acetic, 8 RESULTS AND DISCUSSION Isolation of cecal bacteria from broiler chickens. Initially, 300 anaerobic strains were picked. Some morphologically heterogeneous isolates were then reisolated, thus increasing the total number of strains examined to 325. The percent of the total microflora (direct microscopic counts) cultured in M98-5 medium varied among the six samples from 49.3 to 80.9% (mean of 59.6%). A comparison of different media for the isolation of cecal anaerobes from commercial broilers was part of a previous study (20). Strains were tentatively classified on the basis of morphology, carbohydrate fermentation, fermentation products, and other physiological and biochemical features (Tables 1 and 2). A description of the predominant groups of cecal bacteria isolated follows. With the exception of two groups of facultatively anaerobic cocci and rods (group II and VII, Table 1), the majority of these bacteria were gram-positive and strict anaerobes. Facultatively anaerobic bacteria. Two types of facultative anaerobes were isolated: gram-positive cocci (group II) and gram-negative, motile rods (group VII) comprising 12.6 and 4.9% of the total isolated microflora, respectively. The gram-positive cocci, which were also isolated from chicken cecal contents in a previous study (20), produce lactic acid as a major fermentation product. Under anaerobic conditions they form acid from glucose, fructose, lactose, maltose, and sucrose. All of the grampositive cocci examined (eight strains) hydrolyze gelatin and tributyrin and reduce nitrate to nitrite. Catalase activity could be demonstrated in cultures grown aerobically on plate media but not in cultures grown anaerobically in prereduced media (20). Although these organisms were not identified previously (20), we now believe they may be related to the genus Staphylococcus. Tentative identification is based on the fact that they are facultatively anaerobic and produce acid from glucose anaerobically and aerobically (1). In addition, they produce major amounts of lactic acid from glucose (anaerobically) as do other strains of Staphylococcus (S. aureus and S. epidermidis) we have examined. Facultatively anaerobic, gram-negative rods of group VII were presumptively identified as Escherichia coli in tests with the improved Enterotube (Roche Diagnostics). These bacteria were present in five of the six cecal samples (Table 3) and constituted 2 to 13% of the isolated strains. Axnaerobic cocci. Bacteria in group I were gram-negative, pleomorphic cells with many club, dumbbell and budding forms in pairs and streptococcal-like chains. It is uncertain whether these spherical to elongated cells are truly cocci or rods because of their extreme pleomorphism. We have temporarily designated them as budding cocci. Strains similar to this unnamed species have been isolated from the chicken cecum (4,20), human feces (J. Gossling, Abstr. Annu. Meet. Amer. Soc. Microbiol., p. 81, 1972) and human uterus (12). In a rumen fluid basal medium these bacteria do not ferment sugars but do hydrolyze esculin ( Table 2). Fermentation products from glucose include minor amounts of formic, acetic, and butyric acids and some H2 gas (0 to 2.6%, vol/vol). Strains in group III are large (1.5 to 2.0 tm) gram-positive cocci found in pairs and chains which are probably related to species of Peptostreptococcus Kluyver and Van Niel (19). These anaerobic streptococci differed from Peptostreptococci cultured from chicken cecal contents in a previous study (20) inasmuch as they fermented several sugars. These strains resembled P. intermedius as described by Holdeman and Moore (15) a Media and methods for most tests are given in references. Production of ammonia was determined with Nessler reagent in media used for indole production. Lipolysis was determined in basal media containing 1% (vol/vol) tributyrin (Sigma Chemical Co.) as fermentable substrate. Reactions given are for most strains without a group. Symbols: -, no fermentation (terminal pH 6.3 to 7.0) or no reaction; a, acid reaction (terminal pH 5.5 or less); w, weak reaction (terminal pH 5.6 to 6.2); +, positive reaction; V, variable reaction. Superscripts refer to reactions of a few strains. None of the strains fermented amygdalin or inositol and only 1% of all isolates fermented melezitose. Glycogen and rhamnose were fermented only by group Vc. Motile strains were observed in group VIb. b Designation of amounts of fermentation products is similar to that given in the footnote of Table 1. Products in parentheses are formed by a few strains. strains constituted a small part of the total microflora (Table 1) and were observed in only two out of six cecal samples. Anaerobic gram-positive rods. As in a previous study (20), this group of gram-positive, nonsporeforming rods represented the largest portion of the cecal bacteria isolated. Half of the strains in group IV (18%) were identified as Propionibacterium acnes (IVa). These belonged to serotypes I and II on the basis of sorbitol fermentation (15). P. acnes was also isolated from poultry cecal material by Barnes and Impey (3). Groups IVb and IVc resembled species of Eubacterium as they were anaerobic, gram-positive rods primarily producing lactic and butyric acids from glucose (15). Group IVb strains were oval-shaped cells or rods with known Eubacterium species (15). Group IVc included short and long slender rods (0.5 to 1.0 by 2.5 to 5.0 Mm) in chains. Although these strains appear to be closely related to E. tortuosum described by Holdeman and Moore (15), most of our strains ferment salicin and produce indole. Bacteria in group IVd were gram-positive to gram-variable rods (1.0 by 2.5 to 5.0 um) with rounded ends found in long chains. These bacteria differed from any previously described nonsporeforming, gram-positive anaerobes as they characteristically produced acetic and succinic acids and H2 gas from glucose. They may be related to species of Eubacterium on the basis of presumptive criteria for this genus (15). Similar strains of grampositive, nonsporeforming, succinate-producing bacteria have been isolated by E. M. Barnes from poultry cecal material (personal communication). Anaerobic, gram-negative rods. Group V was composed of gram-negative rods and totaled 18.6% of the isolated microflora. Strains in group Va were variable with respect to the fermentation tests given in Table 2. Representative strains were short and long fusiform-shaped rods (1.5 to 2.5 by 2.5 to 5.0 Mm) as singles, pairs, and chains. Fermentation of fructose, glucose, maltose, sucrose, and trehalose as well as formation of formic and acetic acids as major products from glucose and pyruvate were some predominant characteristics of the strains tested. Morphological features of these organisms and their produsts from glucose were similar to Bacteroides biacutus and Bacteroides clostridiiformis (15). They differed from B. biacutus in that melezitose and lactose were not fermented. Phase contrast microscopy revealed that a few strains appeared to have small spores or vacuoles located centrally and subterminally. It is not known whether these are true spores since none of the strains survived a spore heat test (15). Identification of this group is provisional until characteristics of additional strains are examined. Strains similar to group Va were isolated and identified as B. clostridiiformis from chicken cecal contents (20) and from poultry ceca (2). Group Vb were large, blunt-ended rods (2.0 by 5.0 to 7.5 Mm) occurring mainly as singles and pairs. These species were identified as Bacteroides hypermegas since many fermentation reactions agreed well with those of Holdeman and Moore (15). Our organisms fermented a wide variety of sugars, hydrolyzed tribytyrin, and produced major amounts of propionic and acetic acids from glucose. Strains of B. hypermegas were first described by Harrison and Hansen (14) and later isolated by Goldberg et al. (13) and Barnes and Impey (2) from poultry ceca. Bacteria in Proup Vc appear to be similar to Bacteroides fragilis as described by Holdeman and Moore (15). These gram-negative rods (measuring 0.5 to 1.0 by 1.5 to 3.0 Mm) produced succinic and acetic acids and H2 gas (2.1 to 4.8%, vol/vol) from glucose fermentation. All four isolates we examined differed from known B. fragilis strains in that our isolates converted threonine to propionate. They differed from B. ruminicola in that H2 gas was produced in the culture media (5). Preliminary studies by M. P. Bryant have indicated that most strains of B. fragilis can be distinguished from B. ruminicola on the basis of H2 production (unpublished observations). It is possible, therefore, that the succinate-producing, gram-negative rods isolated in this study may be variants of B. fragilis which can convert threonine to propionate. Barnes and Impey (2) have also isolated B. fragilis from poultry ceca. Anaerobic, sporeforming rods. Group VI comprised 16% of the isolated cecal microflora and consisted of three types of Clostridium species which did not liquefy gelatin. Group VIa was represented by large, nonmotile rods measuring 1.5 to 2.5 by 5.0 Mm (some as long as 20 Mum) with blunt ends and subterminal spores. The two strains tested produced acid from fructose, glucose, and mannose and formed butyric and lactic acids as well as copious amounts of H2 gas (9 to 13%, vol/vol) from glucose. These strains may be similar to C. tyrobutyricum on the basis of sugars fermented, products from glucose (15), and lactate fermentation (7). However, our strains appeared to be nonmotile. Strains in group VIb were pleomorphic, motile rods (1.0 by 2.5 to 5.0,um) with terminal and subterminal spores; some cells were typically fusiform-and spindle-shaped arranged as singles, pairs, and a few chains. These organisms weakly fermented fructose and glucose and formed primarily butyric acid from glucose. These strains were similar to those isolated in a previous study (20). Group VIc consisted of oval-shaped rods (1.5 to 2.0 by 2.5 to 5.0 Mm) with subterminal spores occurring as singles, pairs, or chains. These bacteria were nonreactive in several tests (Table 2), producing minor amounts of butyric acid and H2 gas from glucose but large amounts of butyric acid from lactate fermentation. None of the organisms in groups VIb and VIc could be identified as being similar to known species of Clostridium described by Holdeman Preliminary findings on the growth requirements of anaerobic strains. Our previous study on cecal bacteria (20) indicated that rumen fluid and yeast extract stimulated the growth of many anaerobes. Since the growth requirements of chicken intestinal bacteria are largely unknown, we attempted a survey of some factors which might enhance the growth of several cecal isolates from the various morphological groups (Table 1). These data are shown in Table 3. Preliminary experiments (data not shown) with culture gas phase indicated that good growth was observed with Na2CO,-containing media prepared under CO2 or C021N2 atmosphere. Maximun culture density for most strains was achieved with a medium containing Na2CO,-CO2 buffer. Carbon dioxide may be stimulatory to the growth of most chicken cecal anaerobes; however, more rigorous experiments are needed to establish whether particular species of cecal bacteria have an absolute requirement for CO2. Work by Dehority (11) with ruminal anaerobes has revealed that many strains required or were stimulated by CO2. A simple basal medium consisting of minerals, glucose, Na2CO,-CO2, and cysteine-sulfide did not support the growth of any cecal strain examined. The addition of Trypticase and hemin, in particular, to.this medium stimulated the growth of strains similar to Bacteroides fragilis (group Vc). The growth stimulatory properties of hemin for intestinal anaerobes are well known and have been described for human intestinal isolates of B. fragilis (15,17) and ruminal strains of Bacteroides ruminicola (9,10). When yeast extract was included in the basal medium with Trypticase and hemin, the growth of strains from 8 of 11 groups was enhanced. When a vitamin mixture was added to the basal medium, only strains in group Vb grew (Table 3) as well as one strain each from groups Va and VIa (data not shown). Addition of rumen fluid (20% vol/vol) alone to the basal medium supported the growth of only a few groups (Va, Vb, and VIa). Components in rumen fluid other than volatile fatty acids and hemin appear to be required or highly stimulatory for strains in group I (gram-negative, pleomorphic cocci). Most strains of the various groups of cecal bacteria grew well in basal media supplemented with rumen fluid, Trypticase, hemin, and yeast extract (or a vitamin mixture in place of yeast extract). Hemin could be deleted from media containing rumen fluid without affecting the growth of hemin-stimulated strains (group Vc). In this respect, Caldwell et al. (10) showed that rumen fluid contains heme. Although a mixture of volatile fatty acids (VFA) could partially reproduce the stimulation of groups IVc and Va brought about by rumen fluid, VFA may not be required for the growth of most cecal anaerobes. Liver or cecal extract added to the basal medium only enhanced the growth of group Vb strains (B. hypermegas). These observations are consistent with our previous findings that most chicken cecal bacteria do not require liver or cecal extract for growth (20). The growth of some strains of chicken cecal anaerobes isolated by E. M. Barnes, however, appears to be enhanced by a factor(s) in liver extract (unpublished observations). The data in Table 3 suggest that most anaerobic cecal bacteria could be grown in a Na2CO,-CO2 buffered medium containing minerals, cysteine-sulfide (as reducing agent), carbohydrate energy source, rumen fluid, Trypticase, and yeast extract. The fact that yeast extract or a vitamin mixture is highly stimulatory to many cecal bacteria suggests that chicken intestinal anaerobes may have specific vitamin requirements for growth. The growth stimulation by vitamins such as p-aminobenzoic acid, biotin, folic acid, and vitamin B,2 for the cellulolytic rumen anaerobes, Ruminococcus species, and Bacteroides succinogenes (8,21), has been established. Not much is known about the vitamin requirements of noncellulolytic anaerobes. However, Varel and Bryant (24) have recently observed a B,2 requirement (replaced by methionine) for growth with human isolates of B. fragilis. Compositional variations in the cecal microflora of broilers. In this study (Table 1), 83% of the total isolated microflora (325 strains) of 5-week-old broilers consisted of (1) grampositive, facultative cocci (group II, 12.6%), (2) Propionibacterium acnes and Eubacterium sp. (group IV, 36.1%), (3) Bacteroides clostridiiformis, B. hypermegas, and strains similar to B. fragilis (group V, 18.6%) and (4) Clostridium sp. (group VI, 15.7%). The remaining groups of the flora were made up of gram-negative, budding cocci (group I, 5.2%), Peptostreptococcus (group III, 1.5%) and E. coli (group VII, 4.9%). Table 4 shows the distribution of the various morphological groups and subgroups isolated and indicates that considerable variation exists among individual cecal samples. The cecal profile of microorganisms from each animal suggests that some species are isolated with (or associated with) certain other species. For example, in samples 1, 2, and 3, the lactic acid-producing, facultatively anaerobic cocci (group II) were isolated with large numbers of other gram-positive organisms (primarily group IVa strains) along with relatively smaller numbers of sporeforming rods (group VI) and gram-negative rods (group V). In samples 4, 5, and 6, however, the gram-negative rods in group V were isolated with gram-positive rods (groups IVb, c, and d) and sporeforming rods (group VI). It is interesting to note that B. hypermegas (Vb) and B. fragilis-like strains (Vc) were isolated in high numbers (23.0 and 18.2% of the isolated microflora, respectively) in only two samples (4 and 5). These two species were not isolated in our initial study (20). Results of the previous study (20) on chicken cecal microflora of 5-week-old, "laboratoryreared" birds indicated that species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus sp., Propionibacterium acnes, Eubacterium sp., B. clostridiiformis, and Clostridium sp. were isolated. The types of anaerobes isolated from cecal material in our two studies were similar to those described by Barnes and Impey (2) from poultry ceca. However, the relative proportions of the various bacterial types differed. Few studies dealing with intestinal microflora in nonruminants consider the animal to animal variation when describing the predominant bacterial species isolated from intestinal material. Our data indicate that appreciable variation may exist in the cecal microflora among differ-ent animals (as well as within the same animal) reared under similar growth conditions (environment, food, water). These observations suggest that other factors (host-microbe or microbe-microbe interactions) may have some effect on the occurrence and distribution of the various bacterial species in the cecum. These must be taken into consideration in any study involving the isolation, cultivation, identification, and relative importance of species in a mixed culture habitat such as the chicken cecum.

v3-fos

2018-04-03T00:26:09.803Z

1974-04-10T00:00:00.000Z

11245557

s2

Trisporic Acid Biosynthesis in Blakeslea trispora via Mating Type-specific Precursors* Abstract Separate (+) and (-) mating type cultures of Blakeslea trispora synthesized: (a) labeled trisporic acid B and trisporic acid C when incubated with labeled, partially purified extracts isolated from opposite mating type cultures; (b) unlabeled trisporic acids when incubated with labeled glucose and unlabeled extracts isolated from opposite mating type cultures; and (c) over 100-fold less trisporic acids when incubated with labeled extracts isolated from the same mating type cultures. Thus, separate (+) and (-) cultures synthesize mating type-specific precursors of trisporic acids. SUMMARY Separate (+) and (-) mating type cultures of Blakedea trispora synthesized: (a) labeled trisporic acid B and trisporic acid C when incubated with labeled, partially purified extracts isolated from opposite mating type cultures; (5) unlabeled trisporic acids when incubated with labeled glucose and unlabeled extracts isolated from opposite mating type cultures; and (c) over lOO-fold less trisporic acids when incubated with labeled extracts isolated from the same mating type cultures. Sutter and co-workers (4) demonstrated that separate (+) and (-) cultures of B. trispora synthesized small amounts of trisporic acid B and trisporic acid C when stimulated by a neutral fraction isolated from the culture medium of the opposite mating type culture of B. trispora. In this paper, we demonstrate that the neutral fractions do contain mating type-specific precursors of trisporic acid B and trisporic acid C. Labeled PNFi was prepared as follows. Ten 2-liter flasks, each containing 500 ml of PGT medium, were inoculated with (+) mycelia and incubated on a gyratory shaker at 25" (4). After 44 hours, 25 PCi of [ U-i4C]glucose (118 nmoles) were added to each flask and the incubation was continued for an additional 76 hours. The culture medium was collected and extracted in * This work was supported in part by National Science Foundation Grant GB-20533 from the Metabolic Biology Program. $ Present address, Department of Pharmacology,-University of Witwatersrand. (The culture medium was not adjusted to pH 2 prior to extraction, a step performed in previous work (4). This new procedure resulted in a 90 y0 rather than a 40 o/0 recovery of the trisporic acid-stimulating components from (+) culture medium.) The chloroform extract was evaporated to dryness in DUCUO, and the residue dissolved in 6 ml of ethanol and then purified by Sephadex LH-20 Ch romatography (4). The active fractions from the column contained 950,000 cpm and 2,550 A285 units of PNF. Unlabeled I'NF containing 2,790 A285 units was prepared at the same time. I'NF absorbs ultraviolet radiation maximally at 285 nm. AzB5 units arc the A285 reading of the sample times the dilution factor times the total milliliters of undiluted sample solution. Radioactivity measurements were made in a Beckman LS-230 liquid scintillation system after the sample solvent (ethanol) was evaporated from the counting vial by bubbling with nitrogen gas and after 5 ml of Bray's (7) solution without 1,4-bis[2-(5-phenyloxazolyllbenzene was added. Labeled PNF was added to a 36-hour (-) culture, (+) culture, and a 0.5-liter flask containing 100 ml of uninoculated PGT medium and then incubated for 2.5 hours. After the incubation, the culture media were collected, and the acid fractions were isolated and partially purified by DEAE-Sephadex chromatography (4). Column fractions, in which trisporic acids were eluted if present, were pooled and analyzed for radioactivity and A 325. (Trisporic acids absorb ultraviolet radiation maximally at 325 nm.) The purified acid fraction from the (-) culture, but not the (+) culture, contained both radioactivity (29,280 cpm) and ultraviolet-absorbing material (116 A 325 units) above the levels found in the purified acid fraction from uninoculated PGT medium (Table I). In a control experiment in which unlabeled PNF and labeled glucose had been incubated for 2.5 hours with a ( -) culture, (+) culture, and a flask containing uninoculated PGT medium, the purified acid fractions all exhibited the same low radioactivity (Table I). However, the purified acid fraction from the (-) culture, but not (+) culture, contained ultraviolet-absorbing material (122 Aa units) above the level found in the purified acid fraction from uninoculated PGT medium. Trisporic acid B and trisporic acid C in purified acid fractions were resolved and purified by silica gel thin-layer chromatography (4). Trisporic acid B and trisporic acid C, isolated from the (-) culture incubated with labeled PNF, exhibited specific activities of 250 cpm per A325 unit. These observations demonstrate that both trisporic acid C and trisporic acid B were radioactive and therefore PNF contains mating type-specific precursors of trisporic acids. Radioactive trisporic acid C and trisporic acid B were also isolated from uninoculated PGT medium incubated with labeled PNF, indicating that the PNF preparations contained the trace amounts of trisporic acids which accumulate in the culture medium of 5-day (+) cultures (4). Similar experiments were performed with MNF. The quantity of trisporic acid-stimulating components synthesized by (-) cultures, however, is less than fsO the amount synthesized by (+) cultures (4). Werkman and van den Ende (6) reported that trisporic acids, isolated from (+ / -) culture medium by organic solvent extraction and partially purified by DEAE-Sephadex chromatography, stimulated synthesis of these components in (-) cultures of B. trispora. Therefore, labeled MNF was prepared as follows. Forty flasks, each containing 500 ml of PGT medium, were inoculated with (-) mycelia and incubated. Labeled MNF (320 ASO units) was added to each of five 34hour (+) cultures, (-) cultures, and flasks containing 100 ml of uninoculated PGT medium and then incubated for 5 hours. The purified acid fraction isolated from (+) cultures, but not (-) cultures, contained significant radioactivity (18,700 cpm) and ultraviolet-absorbing material (84 A325 units) above the levels found in the purified acid fraction from uninoculated PGT medium (Table II). In a control experiment in which unlabeled MNF and labeled glucose had been incubated with (+) cultures, (-) cultures, and flasks containing uninoculated PGT medium, the purified acid fractions all exhibited the same low radioactivity (Table II). However, the purified acid fraction from (+) cultures, but not (-) cultures, contained ultraviolet-absorbing material (91 AS25 units) above the level found in the purified acid fraction from uninoculated PGT medium. Trisporic acid B and trisporic acid C, isolated from the purified acid fraction of (+) cultures incubated with labeled MNF, represented 65y0 of the A 325 units recovered from the silica gel chromat.ogram. The remaining 35% was distributed between two unidentified compounds which migrated below trisporic acid C. Each trisporic acid exhibited a specific activity of 180 cpm per A325 unit. When the labeled trisporic acid C (11 As25 units) and authentic unlabeled trisporic acid C (99 A 325 units) were co-chromatographed on a Sephadex LH-20 column, the radioactivity and A325 were coincident. A similar coincidence was observed when labeled trisporic acid B (3 A325 units) and authentic unlabeled trisporic acid B (27 A 325 units) were co-chromatographed. These observations demonstrate that both trisporic acid C and trisporic acid B were radioactive and therefore MNF contains mating type-specific precursors of trisporic acids. The experiments reported in this communication differ from the tracer studies of Werkman and van den Ende (6) in three ways. (a) The final radioactivity measurements were made with pure trisporic acid C and trisporic acid B rather than with a partially purified extract of trisporic acids. (b) The radioactivity in the neutral fractions was shown to be incorporated into trisporic acids in a mating type-specific fashion. (c) The labeled compounds in a neutral fraction were shown not to be degraded to metabolites of glucose prior to incorporation into trisporic acids.

v3-fos

2018-04-03T02:09:14.389Z

1974-03-01T00:00:00.000Z

22133046

s2

Comparison of Six Methods for Isolating Mycobacteria from Swine Lymph Nodes Six laboratory methods were compared for isolating acid-fast bacteria. Tuberculous lymph nodes from each of 48 swine as identified by federal meat inspectors were processed by each of the methods. Treated tissue suspensions were inoculated onto each of eight media which were observed at 7-day intervals for 9 weeks. There were no statistically significant differences between the number of Mycobacterium avium complex bacteria isolated by each of the six methods. Rapid tissue preparation methods involving treatment with 2% sodium hydroxide or treatment with 0.2% zephiran required only one-third to one-fourth the processing time as a standard method. There were small differences in the amount of contamination among the six methods, but no detectable differences in the time of first appearance of M. avium complex colonies. Certain routine tissues (1 by their growth on Lowenstein-Jensen media containing 5% sodium chlo-ride at 28 days (5). Serological identification of the mycobacteria was done by the agglutination method of Schaefer (7). Certain routine mycobacteriological examination methods require elaborate and expensive equipment and are laborious. An increased interest in obtaining rapid diagnostic methods for isolating mycobacteria from animal tissues has resulted from recent changes in Meat and Poultry Inspection Program regulations for handling swine carcasses suspected of being tuberculous. In this report, six different laboratory methods were compared for isolating mycobacteria from tuberculous swine lymph nodes identified by federal meat inspectors at an abattoir in Nebraska. MATERIALS AND METHODS Tuberculous lesions in cervical and mesenteric lymph nodes were identified by federal meat inspectors. These were removed entirely or in part from swine carcasses and placed in jars containing a saturated solution of sodium borate for transportation to the laboratory. Upon arrival at the laboratory, each specimen was removed from the sodiurt borate solution; the fat was removed with sterile instruments and the node was placed in a dilute hypochlorite solution (0.1% NaOCl, wt/vol) for 16 h to control surface contamination. The following six methods of decontamination were used for preparing tissue suspensions. Direct inoculation (DI). About 0.2 g of the suspected tuberculous lymph node was removed and submerged in 1.5 ml of 0.2% zephiran for 15 min; it was then transferred to a petri dish and crushed with sterile forceps, and 1.0 ml of nutrient broth was added. Two drops of the tissue suspension was used to inoculate each medium slant. The remainder of the lymph node was ground in a tightly closed screw-cap blender jar for 2 min, and then 50 ml of nutrient broth (Difco no. 0003-01, Detroit, Mich.) containing 0.4% phenol red indicator was added. Samples of the remaining tissue suspension were processed as follows. NaOH. Equal volumes of 2% sodium hydroxide and ground tissue suspension (1.5 ml of each) were placed in a sterile screw-cap centrifuge tube (20 by 125 mm) and allowed to stand for 20 min. The suspension was neutralized by adding 1.25 N hydrochloric acid, mixed by hand shaking, and then allowed to sediment for 5 min. Media were then inoculated with 0.2 ml of the supernatant fluid. NaOH plus centrifugation. This method included the treatment utilized in the NaOH method plus centrifugation of the neutralized suspension at 2,000 rpm in an international model UV centrifuge with a no. 266 rotor (1,000 x g) for 15 min. The supernatant fluid was discarded, and the sediment was suspended in 1 ml of nutrient broth. The suspension (0.2 ml) was used to inoculate each medium slant. Zephiran treatment (Z). Equal volumes (1.5 ml of each) of ground tissue suspension and 0.2% zephiran solution were placed in a test tube (20 by 125 mm), mixed thoroughly by hand shaking, and allowed to stand for 15 min. Media were inoculated by using 0.2 ml of' material from the central portion of this tissue suspension. Papain-zephiran (PZ). Papain (100 ml; Nutritional Biochemicals Corp., Cleveland, Ohio) was added to the remaining tissue suspension of about 45 ml. Sufficient sodium hydroxide was added to change the indicator from yellow to pink. The suspension with a Teflon stirring bar was placed on a magnetic stirrer at 23 C for 30 min. Equal volumes of this suspension and 0.2% zephiran (1.5 ml of each) were mixed and allowed to stand for 15 min. The suspension was thoroughly mixed, and each medium slant was inoculated by using about 0.2 ml of the suspension. Papain-pentane-zephiran (PPZ) (6). The remaining tissue suspension prepared for PZ treatment was allowed to digest for an additional 30 min, 10 ml of pentane was added, and the contents were .mixed by hand and allowed to stand for 30 min. The pentane layer was removed with a sterile pipette and filtered through sterile muslin in a funnel. The filtrate was then centrifuged for 20 min at 1,000 x g. Nutrient broth (2 ml) was added to the sediment and the contents were shaken vigorously. Equal volumes of 0.2% zephiran and the suspension were mixed and allowed to stand for 15 min. Each medium slant was inoculated with about 0.2 ml for the suspension. Eight medium slants, including two tubes of Lowenstein-Jensen medium, two tubes of Stonebrink medium, one tube of Herrold egg yolk medium, one tube of Herrold egg yolk medium with malachite green, one tube of Herrold egg yolk medium with mycobactin (2 ug/ml), and one tube of Middlebrook 7H10 medium were inoculated with material prepared by each of the tissue preparation methods. The media were incubated at 37 C and examined at 7-day intervals for 9 weeks. Smears of colonies were stained with carbolfuchsin for detection of acid-fast bacilli. RESULTS The results of mycobacteriological examinations are presented in Table 1. Colonies were initially rounded and white and usually appeared buff colored after 5 weeks of growth. Cultures of slowly growing nonphotochromogenic mycobacteria were isolated from 36 of the 48 specimens. These cultures were subsequently identified as Mycobacterium avium complex bacteria (8,9) by their resistance to chemical compounds. Serological tests on these 36 strains revealed that 12 were serotype 1, 19 were serotype 2, 3 were serotype 3, 1 was serotype 13, and 1 was serotype 17. Rapidly growing mycobacteria were isolated from 8 of the 48 specimens from which no other acid-fast bacilli were isolated. A comparison of the efficacy of each of the six methods for isolating M. avium complex from swine lymph nodes and the time required for processing specimens by each of the methods are shown in Table 2. The number of M. avium complex isolations made varied from 31 to 34 for each of the six methods. These numbers were not significantly different as determined by the chi-square test (P = 0.75). In no instance were all 36 of the M. atium complex isolations made by any one method. The time required for processing tissue specimens by each of three rapid methods in order of decreasing efficiency was: (i) DI, (ii) Z, and (iii) NaOH. The time saved by the rapid methods as compared to the PPZ method was 72% for the DI method, 66% for Z method, and 60% by the NaOH method. As a group, the three most rapid methods (NaOH, Z, and DI) had significantly fewer contaminated media slants than tissue suspensions prepared by the PPZ method ( Table 3). The computed chi-square value (1 degree of freedom) was 9.49, indicating that a real difference was highly probable. Herrold egg yolk medium slants were generally contaminated 'Total number of trials was 1,728. LJ, Lowenstein-Jensen medium; ST, Stonebrink medium; 7H10, Middlebrook 7H10 medium; Hy, Herrold egg yolk medium; Hg, Herrold egg yolk medium plus malachite green; Hm, Herrold egg yolk medium plus malachite green plus mycobactin. 'Mean value for two tubes. more frequently than other media, but considerable variation among all media was apparent. Isolation failures for the different culture media and processing methods are presented in Table 4. Isolation failures occurred more frequently on Middlebrook 7H10 medium than on the other media. The computed chi-square value was 20.8, which can occur by chance less than 5 times per 1,000. Isolation failures among the other five methods fluctuate within chance variation. The largest number of M. avium complex isolation failures (47/66) recorded on 7H10 medium was for specimens treated with zephiran. Isolation failures were greater for the PZ method than fox the other five methods. However, there was not sufficient evidence in these data to establish a true difference among the six methods studied. The time of first appearance of the M. avium complex colonies ranged from 2 to 9 weeks for the six different methods (Table 5). Five weeks after inoculation, 97% of the specimens treated by the NaOH method were culture positive, 93% of the specimens treated with the Z method were positive, and 89% of the specimens processed by the DI method were positive. The observed differences in colony appearance at 5 weeks are within the limits of random variation, as determined by a chi-square test. DISCUSSION The rapid methods described in this report provide for a marked reduction in processing time, varying from 42 to 72% of that of PPZ method currently in use in this laboratory. These benefits were achieved with no detectable increase in contamination or significant reduc-tion in isolation rate. The mycobacterial isolation rates from tuberculous swine lymph nodes for each of the six different processing methods were in close agreement with reports using other accepted procedures (1-3). Differences have been reported between the total number of mycobacterial isolations obtained on egg medium as compared to agar medium when mycobacteriological specimens are treated with zephiran (4). Isolation failures on agar medium have been attributed to a carry-over of traces of zephiran in the inoculum which is inactivated by the lecithin present in egg medium. The data presented herein comparing the number of M. avium complex isolation failures on Middlebrook 7H10 media with those observed on egg containing media support this finding. Only small amounts of tissue (0.2 g) or tissue suspension (1.5 ml) were processed by rapid methods in comparison to the amount of tissue suspension (40 ml) used in the PPZ method, yet the isolation rates for the rapid methods were not significantly decreased as compared to the rate for the PPZ method. Downloaded from It is apparent from the data obtained in this study that a substantial financial savings can be realized by using one of the rapid techniques. Furthermore, it should be emphasized that the rapid methods described may be implemented in a diagnostic laboratory without the addition of expensive equipment.

v3-fos

2018-04-03T01:33:56.859Z

1974-09-01T00:00:00.000Z

29874658

s2

Development of a Selective Enterococcus Medium Based on Manganese Ion Deficiency, Sodium Azide, and Alkaline pH Rogosa broth, without its salt supplement and dissolved in deionized water, was adapted for the selective isolation and enumeration of enterococci. This medium supported good growth of enterococci, but it suppressed growth of other lactic acid bacteria. The sensitivity and specificity of the medium were tested after addition of various increasing concentrations of NaN(3) against known strains of enterococci and other bacteria. Many strains of Streptococcus faecium showed low azide tolerance; optimal growth was obtained at a concentration of 0.01% NaN(3), which totally or partially inhibited unrelated species of lactic acid bacteria. The selectivity of the medium was further increased by pH adjustment to 9.6. Carbonate and Tween 80 were added to overcome partial inhibition of enterococcal growth by the new combination of selective conditions. The final medium was evaluated in agar form in isolations from human and animal feces, polluted water, meat, and dairy products. Counts were obtained after 16 to 17 h of incubation at 37 C. The isolates satisfactorily conformed to the group characteristics of enterococci. Depletion of available Mn2+ levels in nutrient media results in a significant reduction of growth by lactobacilli (7) and pediococci (6), but has no effect on the growth of enterococci (6). Since the former groups of bacteria frequently occur in nature together with enterococci, the utilization of Mn2+ deficiency might facilitate the isolation of enterococci from mixed microfloras. A selective medium utilizing this premise was developed in the present study. The sensitivity and specificity of the medium was tested against known strains of bacteria and in isolations from various natural sources. (This investigation was presented in part at the 73rd Annual Meeting of the American Society for Microbiology, Miami Beach, Florida, 6-11 May, 1973.) MATERIALS AND METHODS Cultures. Authenticated strains of enterococci were used for the initial evaluation of the medium. They comprised Streptococcus faecalis, American Type Culture Collection (ATCC) 11700, 14428, and strain 19-1, obtained from W. H. Seeley, Cornell University; S. faecium typical strains, ATCC 6057, and 13 other strains from the culture collection maintained in this laboratory. The latter strains had been previously isolated from dairy products and identified according to Deibel (3,4); S. faecium atypical strains, FCMA-2 (var. casseliflavus) and FCMA-11, were obtained from J. 0. Mundt, Univer-sity of Tennessee, and T-15 (a Langston strain isolated from grass silage) was received from H. W. Seeley. In addition, we included three strains of S. bovis, SS-752, 963, and 964, and three strains of S. equinus, SS-945, 946, and 947, received from R. R. Facklam, Center for Disease Control, Atlanta, Ga. Other lactic acid bacteria, not related to fecal streptococci, included: S. lactis ATCC 7962, S. cremoris ATCC 9625, S. diacetilactis DRC-1, and two group N strains received from Rebecca Lancefield, Rockefeller University, New York; Leuconostoc mesenteroides, two strains obtained from J. 0. Mundt, L. dextranicum Ld688 and L. citrovorum 91404, obtained from W. E. Sandine, Oregon State University, and L. citrovorum Al, obtained from T. W. Keenan, Purdue University; Pediococcus cerevisiae ATCC 8081, 8042, 10981 and strain no. 25, obtained from H. W. Seeley; Lactobacillus bulgaricus G5 and L. jugurti G2, isolated from Greek yogurt by C. J. Efthymiou; and one strain each of L. casei and L. brevis, obtained from the Midwestern Culture Service, Terre Haute, Ind. Five strains of different gram-negative and gram-positive organisms with cytochromeand catalase-dependent metabolism (see Table 3) were also included to test the selective capacity of the NaN,-containing medium. Culture media. As basal medium we used a modified Rogosa broth (6); the salt supplement of the complete medium was deleted; all ingredients were dissolved in deionized, all-glass-distilled water. NaNs was added at different, increasing amounts: 0.01 0.015, 0.02, 0.025, 0.03, and 0.05%. The resultant media were evaluated against our test cultures. The concentration adopted for the final medium was examined, and we finally selected pH 9.6 as the most suitable; in conjunction with this modification, 5.3 g of Na2CO, per liter was added. The final selective medium of this study (Table 1) was prepared in the following manner: Trypticase, yeast extract, tryptose, ammonium citrate, Tween 80, sodium acetate, and glucose were dissolved in 150 ml of water; the K2HPO4 salt was dissolved in 50 ml of water; the carbonate was dissolved in 50 ml of water and was autoclaved in a screw-capped bottle with its cap tightened. All three solutions, autoclaved for 15 min at 15 lb/in2 (121 C), were mixed aseptically at room temperature, and the pH was adjusted to 9.6 with sterile 2 N NaOH. Five milliliters of 2% Seitz-filtered NaN, was added. The temperature of the medium was then raised to 70 C in a water bath, and a sterile solution of agar (15 g dissolved in 740 ml of water) was added and mixed well. After mixing, the pH was rechecked and, if necessary, readjusted. Avoiding direct exposure to sunlight, the medium was poured into plates, allowed to solidify, and stored in a refrigerator. The medium was also tested as broth (agar omitted). All dissolved ingredients making up the broth were mixed aseptically at room temperature. For application, a sterile membrane filter (pore size 0.22 gm) was aseptically placed in a sterile filter holder. Ten milliliters of each cheese solution tested was introduced into the funnel and vacuum was applied. The funnel wall was rinsed with 50 ml of sterile phosphate buffer (pH 7.2). After filtration, the filter was aseptically transferred to a plastic petri dish containing an absorbent pad already saturated with about 2 ml of broth, and incubated. Enterococcus counts of various natural samples were determined on this medium. Total viable counts of the same samples were obtained on standard methods agar (BBL). Culture methods. The selective medium was tested in both liquid and agar forms. Inocula of the test organisms were prepared by culturing each strain in basal broth. One drop of a 24-h culture was added from a sterile Pasteur pipette to each tube containing 8 ml of medium. In those cases where gram-negative organisms were tested (Table 3), the inocula were alternatively prepared by diluting the 24-h cultures 1:100 in phosphate-buffered saline (pH 7.0). For the Water, all-glass-distilled and deionized, to 1,000 ml (pH adjusted to 9.6) inoculation of agar plates, one drop of culture was spread in a standard three-way streak on the surface of the medium; the plates were incubated aerobically. All test media were incubated at the optimal temperature for each species tested. All enterococcus cultures and the isolation plates inoculated with natural specimens were incubated at 37 C, except for some tests, which, as indicated, were incubated at 45 C. Isolation of enterococci from natural sources. Fecal samples from cattle, sheep, swine, chickens, and ducks were obtained at the farm of the State University of New York, Farmingdale, N.Y. Human and dog fecal specimens were also used. Freshly voided samples were taken in sterile bottles and refrigerated until analysis. Five-gram samples were weighed, transferred aseptically into 100 ml of sterile phosphate-buffered saline (pH 7.0), dispersed for 45 s in a Waring blender, and then serially diluted. Portions of 0.1 ml were spread on the surface of agar plates with a bent glass rod. Water samples were taken from rivers, creeks, and nearshore points in the environs of New York City and Long Island. Volumes of 50 ml were centrifuged at high speed, the supernatants were siphoned off, and the sediments were taken up in 2 ml of sterile distilled water. Portions were deposited on agar plates and spread as described. Food samples included meat sausages and three cheese varieties purchased at a local New York market. The preparation of these samples for analysis followed established procedures (9,13). After enumeration at 24 and 48 h, colonies from all specimens cultured on the selective agar were picked and seeded in basal broth. The isolates were examined for conformity to the characteristics of enterococci (Sherman's criteria). They were also checked for catalase production and for group D antigen. The latter test was carried out microscopically by using a fluorescein-conjugated, group D-specific antiserum (Sylvana). RESULTS The addition of increasing concentrations of NaN3 to the nutrient base afforded an initial evaluation of sensitivity. Table 2 presents the effect of such additions of NaN3, on the growth of 20 enterococci and 18 lactic acid bacteria. At a NaN3 level of 0.02%, S. faecium grew weakly or not at all; when the azide level was decreased to 0.015%, most typical S. faecium strains grew well, and at 0.01% azide, even atypical variants isolated from plants grew well. S. faecalis, as expected, exhibited greater tolerance toward NaN,, as all strains grew at concentrations 0.02 to 0.05%. Certain of the tested lactobacilli, pediococci, and lactic streptococci showed equal or greater resistance than S. faecium to NaN3. The basal medium, supplemented with 0.01% NaN,, provided a maximum limit for satisfactory growth of the typical and atypical strains of S. faecium ( Fig. 1 and 2), but 0.01% NaN3 was insufficient to completely inhibit other lactic 412 APPL. MICROBIOL. on March 23, 2020 by guest http://aem.asm.org/ Downloaded from cocci and bacilli. Incubation at 45 C, tried as a supplementary selective factor, proved unsatisfactory, since some of our enterococci failed to grow within 24 h and, in some cases, even 48 h. All enterococci grew well in the basal medium adjusted to pH 9.6, whether in the presence or absence of 0.01% NaN, ( Table 3). The high alkaline pH inhibited growth in all six strains of non-enterococcal group D streptococci. To ascertain the alkaline growth limits of these strains, we varied the pH of the medium and adjusted it to lower values. We found that they could grow well at pH 8.9. The 10 strains of lactic cocci grew slowly in the alkaline, NaN,free medium within a 72-h period; only half of these strains could show some growth in the alkaline medium supplemented with 0.01% NaN. The pediococci and lactobacilli were unable to produce visible growth even after 72 h, either in the alkaline, NaN,,-free medium, or in the alkaline, 0.01% NaN,-containing medium. The five cytochromeand catalase-containing, gram-positive or gram-negative strains demonstrated a slight retardation of growth in the alkaline, NaN,-free medium, but they showed a marked retardation of growth and no visible growth before 24 h of incubation in the alkaline medium containing 0.01% NaN. This partial inhibition became more distinct when we used diluted (1:100) inocula. On the alkaline agar medium (pH 9.6 plus 0.01% NaN3), the enterococcus counts from cured meat, cheese, water, and feces (Table 4) varied between less than one enterococcus per ml of water to 20 x 108 per gram of sheep feces. The ratios of enterococci to the total count also varied; in the chicken feces, the enterococci were nearly as numerous as the total count, whereas in the dog feces the ratio was 1:60,000. Colonies of gram-negative bacteria that predominated in most of the fecal specimens, when grown on standard methods agar, were effectively suppressed by the selective medium ( Medium on the left contains 0% NaN, (control). 3). On the standard methods agar, the food samples yielded mostly small or pinpoint-size colonies, typical of gram-positive bacteria. On the selective medium, the colonies exhibited uniform morphology, whereas microscopically, they yielded gram-positive cocci. Upon prolonged incubation, i.e., 48 to 72 h, on some of the selective agar plates that were seeded with cheese samples, some hazy growth appeared around the well-developed enterococcus colonies (Fig. 4). Microscopic examinations revealed pleomorphic gram-positive bacilli. The medium used in these experiments was prepared with common-grade bacteriological agar. When purified, demineralized agar (Oxoid, Ion Agar no. 2) was substituted, the secondary growth was not observed. The observation indi-cates that inorganic ion depletion enhances the selectivity of the enterococcus medium. The round surface colonies obtained on the selective medium reached an average diameter of 1 to 2 mm after 16 to 17 h of incubation at 37 C. One hundred ninety-four colonies isolated at random from the 14 analyzed specimens (Table 4) conformed substantially to the Sherman criteria for enterococci; few possessed catalase, and they reacted well with group D, fluorescein-labeled antiserum. Among 151 isolates from the fecal and water samples, there were six catalase-positive strains. Such strains were not encountered at all among the isolates from the fermented meat and dairy products. Thirtyeight out of 42 strains, tested with the group D antiserum, produced reactions of a 2+ or higher intensity, whereas four showed a weaker reaction or no reaction at all. A few isolates showed nonconformity with one or another of the Sher-, man characteristics. Lack of agreement in more than one of the Sherman characteristics was observed in few of these variants. DISCUSSION About 20 years ago, Reinhold et al. (8) developed a selective medium containing 20 g of sodium citrate per liter and 0.01% NaN3. At this concentration, the NaN, had generally no adverse effect upon the numbers of enterococcus colonies that developed on the agar plates, although a slight inhibition of some unidentified enterococci was noticed. In higher concentrations (0.015 to 0.025%) a decrease in colony size was noted. At 0.03% NaN3, both the size and number of colonies were markedly inhibited. According to Reinhold et al. the 0.01% level of NaN8 showed highest sensitivity, not only in terms of recovery rates, but also in readability of colonies on the agar plates. We adopted the 0.01% concentration of NaN8 as the desired level of chemical inhibitor for our basal medium because of its least inhibitory effect on the growth and colony development of diverse forms of enterococci (Table 2, Fig. 1 and 2). We included a moderate concentration (2 g/liter) of citrate; as an energy source, we primarily used glucose. For a suppression of lactobacilli and physiologically related bacteria, we depended in part on Mn2+ depletion (5, 7). Adjustment of pH to 9.5 to 9.6 was utilized originally by Sherman and Stark (10) as a tolerance test for the identification of S. faecalis, and later by Smith and Sherman (11)and others as a general differential characteristic of the entire group of enterococci. Certain forms of enterococci, however, grew poorly or not at all at this pH, especially after short incubation periods. Chesbro and Evans (2) studied the factors affecting the growth of enterococci in highly alkaline media. They found that the addition of carbonate and oleate (Tween 80) markedly stimulated the growth of enterococci in media with a pH higher than 9. When our basal medium was supplemented with carbonate, the high alkalinity of the modified medium (pH 9.6) did not interfere with the growth of any of the 20 strains tested (Table 3). It is noted that Burkwall and Hartman (1) found that the addition of (12) observed that 0.01% NaN, in blood agar was the critical concentration for prevention of spreading growth of Proteus sp. and coliform bacteria, since a 0.008% level of NaN5 allowed such growth to occur. The delayed growth of the cytochrome-possessing strains in our alkaline plus 0.01% NaN.-containing medium suggests that the nutrients of this medium may favor a certain degree of bacterial resistance. Raising the level of-NaN, to higher than 0.01% levels would probably eliminate the growth of these strains. Nevertheless, we did not attempt such an increase since we intended to maintain the established sensitivity for enterococci at the 0.01% level, and also because isolations from fecal samples (Fig. 3, Table 4) indicated an efficient, for all practical purposes, suppression of gram-negative bacilli. In the isolations of enterococci from various natural habitats (Table 4), the enterococcus counts were determined within 16 to 17 h of incubation. This early enumeration proved useful from the selection point of view. Bacteria unrelated to enterococci that were able to grow on the experimental medium slowly or marginally (Table 3, Fig. 4) did not interfere with the presumptive count and isolation of the enterococci. Partial identification of the isolated strains disclosed an overwhelming proportion of confirmed enterococci ( Table 4). The environmental stress, to which enterococci are subjected in nature and particularly in foods, requires optimal growth conditions during isolation. Our results indicate that such optimal conditions needed for cell resuscitation may be very important for biotypes of enterococci with low tolerance toward the selective agents used in the isolations. The initial evaluation of the medium indicates a favorable performance on this essential point. A comparison of the developed medium with several standard enterococcus selective media is the subject of another report (5).

v3-fos

2018-04-03T05:01:22.311Z

1974-04-01T00:00:00.000Z

25994753

s2

Relationship of cellular fatty acid composition to survival of Lactobacillus bulgaricus in liquid nitrogen. Concentrated cultures of Lactobacillus bulgaricus were prepared by resuspending cells grown in semisynthetic media in sterile 10% non-fat milk solids. The concentrated cultures were frozen in liquid nitrogen for 24 h. The cell suspensions exhibited decreased viability after storage, and the amount of death varied among the different strains tested. Storage stability of all strains examined was improved by supplementing the growth medium with sodium oleate. Radioisotopes were used to study the fate of sodium oleate with L. bulgaricus NCS1. [1-(14)C]sodium oleate was incorporated solely into the lipid portion of the cells, including both neutral and polar lipids. The fatty acid composition of L. bulgaricus NCS1, NCS2, NCS3, and NCS4 grown with and without sodium oleate was studied. The major fatty acids of strains NCS1, NCS2, and NCS3 grown without sodium oleate were dodecanoic, tetradecanoic, hexadecanoic, hexadecenoic, and octadecenoic acids. In addition to these, strain NCS4 contained C(19) cyclopropane fatty acid. The major fatty acids of all strains grown with sodium oleate were tetradecanoic, hexadecanoic, hexadecenoic, octadecenoic, and C(19) cyclopropane fatty acids. All strains grown in broth containing sodium oleate contained larger amounts of octadecenoic and C(19) cyclopropane fatty acid, and less saturated fatty acids than when grown without sodium oleate. Statistical analyses indicated that C(19) cyclopropane fatty acid was most closely related to stability of the lactobacilli in liquid nitrogen. A negative regression line that was significant at P < 0.001 was obtained when the cellular content of this fatty acid was plotted against death. Previous work from this laboratory (25) has shown that cells of Lactobacillus bulgaricus grown in media containing Tween 80 (polyoxyethylene sorbitan monooleate) were more resistant to freezing than those grown without it. Although such an effect had not been previously reported for the lactobacilli, it has been well documented that non-ionic detergents containing oleic acid, and free oleic and cis-vaccenic acids are important in the metabolism of lactobacilli (22,29,30). Either oleic or cis-vaccenic acids can replace the requirement for biotin by the lactobacilli (4,10,29,30). Tween 80, which is a non-toxic form of oleic acid, can also replace this requirement (3, 29, :0). In addition, these acids are intimately involved in the control of the fatty acid synthesis in Lactobacillus plantarum (1,9,28). Oleic and cis-vaccenic acids are incorporated intact into the lipids of lactobacilli or converted to a C1, cyclopropane fatty acid (17,21). C,, I Paper no. 4145 of the Journal Series of the North Carolina State Univ. Agricultural Experiment Station, Raleigh, N. C. cyclopropane fatty acids are formed from oleic or cis-vaccenic acid by the addition of a methylene bridge carbon across the double bond. S-adenosylmethionine serves as the donor of the bridge carbon for this conversion (22). Although C1, cyclopropane fatty acid(s) are found in the lipids of many different types of bacteria, their exact physiological function is unknown. It is believed to have a role in maintaining cell membrane flexibility (16). This study was initiated to determine the mechanism(s) whereby Tween 80 imparted freezing stability to L. bulgaricus. MATERIALS AND METHODS Cultures. The strains of L. bulgaricus selected for this study are used commercially in the manufacture of yogurt and Italian cheese. They were propagated and stored as described previously (25). A medium containing 2% Tryptone (Difco), 1% yeast extract (BBL), 2% Preparation of concentrated cell suspension. Each strain of L. bulgaricus was grown in the control broth and was used to inoculate the test growth media using a 2% inoculum. The cultures were grown statically to the stationary phase (15 h) in the test media at 37 C. They were harvested, concentrated, and stored in liquid nitrogen, and viability was measured as described previously (25). Incorporation of [1_14C]sodium oleate. Cells from 200 ml of broth containing [1-4C Isodium oleate and from 200 ml of broth containing unlabeled sodium oleate were washed three times with cold distilled water at a ratio of approximately 250 mg of cells (dry weight) to 20 ml of water. The cells were recovered between washings by centrifugation at 0 C and 17,300 x g for 10 min. After the final wash the cells from each broth were suspended in 10 ml distilled water, and the resulting suspensions were combined and stored in a freezer until analyzed. The spent medium, washings, and cells were analyzed for 14C content by using a Packard model 574 Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.) to ascertain whether or not sodium oleate was incorporated into (or adsorbed onto) the cells during growth. The counting fluid contained Triton X-100, 333 ml; toluene, 167 ml; 1,4-bis[2(5-phenyloxazolyl)]-benzene, 0.1 g; and 2,5 diphenyloxazole, 5.5 g. Ten ml of the counting fluid was added to each sample, and the final volume was adjusted to 20 ml with toluene. Whole cells were kept in suspension with the aid of Thixotropic Gel Powder (Packard Instrument Co., Inc.). Counts were corrected for background and quenching. Free lipids. Free lipids were extracted from washed whole cells by the method of Bligh and Dyer (2). The cells were extracted three times at 4 C. One part chloroform and 2 parts methanol were mixed with 0.8 parts of an aqueous cell suspension. The first extraction was for 1 h and 40 min, and the second and third were for 30 min each. After each allotted extraction time, 1 part chloroform and 1 part distilled water were added. The mixture was shaken and set aside for 20 min to allow the phases to separate. Between extractions the cells were recovered by centrifugation at 0 C and 17,300 x g for 10 min. The three extracts were pooled and condensed for analyses. Bound lipids. Cell suspensions from which free lipids had been extracted were hydrolyzed for 1.5 h in 1 N HCl at 121 C. The hydrolysates were extracted for 10 min by using the Bligh and Dyer procedure (2). Whole cells (not previously extracted) were also hydrolyzed and extracted for 10 min to obtain information for comparing total and bound lipids. Silicic acid column chromatography. Free lipids were separated by silicic acid chromatography into neutral and polar lipid fractions by using procedures outlined by Dittmer and Wells (6). Gravimetric analysis. Lipid fractions were evaporated to dryness by using a rotary flash evaporator at 42 C. They were redissolved in chloroform and quantitatively transferred to a tared evaporating flask. The chloroform was removed by evaporation, and the flask was dried and placed in a desiccator for at least 30 min before weighing. Gas-liquid chromatography. The fatty acid methyl esters were prepared from the lipid fractions by the method of Metcalfe et al. (20) using boron trifluoride (BF,)-methanol reagent (Fisher Scientific Co.). The methyl esters were separated on a Packard 800 series gas chromatograph (Packard Instrument Co., Inc.) equipped with a flame ionization detector. A stainless-steel column packed with EGSSX (10%) on Chromosorb Q (100/120 mesh, Applied Science Co., State College, Pa.) was employed. The oven temperature was held at 140 C for 3 min after sample injection and then increased linearly at 4 C per min until the final temperature of 190 C was reached. The injector and detector temperatures were 230 and 205 C, respectively. Nitrogen served as the carrier gas with a flow rate of 20 ml/min at 22 C. The fatty acid methyl esters were tentatively identified by comparing their retention times with known standards. The peak areas were determined by triangulation, and the total area was used to determine the relative percent of each fatty acid present. Gas chromatographic detector response was linear over the range studied. Statistical analysis. The regression of percent death on percent fatty acid content was determined by using methods outlined by Snedecor and Cochran (26). RESULTS Relationship of sodium oleate to storage stability. Previous work in this laboratory (25) demonstrated that cells of L. bulgaricus grown in broth containing Tween 80 were more resistant to freezing in liquid nitrogen than cells grown in broth without Tween 80. When most of the free oleic acid associated with Tween 80 was removed by silicic acid column chromatography, the detergent lost part of the ability to impart freezing resistance to cells grown in its presence. Sodium oleate added to a broth composed of Tryptone, yeast extract, and lactose was toxic to the growth of L. bulgaricus. However, Tween 20 (polyoxyethylene sorbitan monolaurate) was added to the medium to detoxify the fatty acid (15,30). Cells grown in the control broth without sodium oleate exhibited susceptibility to freezing similar to that observed in a previous report (25). An improvement in the storage stability was noted for each strain when grown in the presence of sodium oleate ( Table 1). The cultures grown without sodium oleate varied in their resistance to freezing. Strain NCS4 was most resistant to freezing; strain NCS1 was most sensitive and exhibited the greatest response to sodium oleate. Although the level of sodium oleate utilized in these experiments improved the stability of strains NCS2 and NCS3, it did not impart to the cells sufficient resistance to survive the freezing completely. Previous studies in our laboratories (25) indicated that strains of L. bulgaricus varied with respect to the optimum level of Tween 80 required to produce cells that were resistant to freezing. Presumably a similar situation exists with respect to the optimum level of sodium oleate. Incorporation of [l-14C]sodium oleate. Radioactive tracer studies demonstrated that sodium oleate was incorporated into the lipid portion of L. bulgaricus NCS1 ( Table 2). Analysis of whole cells revealed that 100.8% of the radioactivity associated with the cells was incorporated as lipid material of which 28.5% was bound lipid and 72.3% was free lipid. The free lipid was further separated by silicic acid col- umn chromatography into neutral and polar lipid fractions. There was approximately 2.5 times more 14C associated with the polar lipids than with the neutral lipids. Gravimetric analysis of the lipids revealed that the percentage by weight of each fraction was similar to the percent distribution of radioactivity in each fraction. There was little quantitative difference in lipid content between cells grown with and without sodium oleate. The total amount of lipid for cells grown with and without sodium oleate was 4.2 and 4.9%, respectively. Fatty acid analysis of lipids from hydrolyzed whole cells. Lipids from L. bulgaricus NCS1, NCS2, NCS3, and NCS4 grown with and without sodium oleate were evaluated for fatty acid composition. Chromatograms of fatty acid methyl esters prepared from lipids extracted from hydrolyzed cells of strain NCS1 grown with and without sodium oleate are presented in Fig. 1. Similar chromatograms were obtained for strains NCS2, NCS3, and NCS4. Quantitative evaluations comparing percentage compositions based on peak areas for all four strains are presented in Table 3. The primary fatty acids in lipids from hydrolyzed whole cells grown in sodium oleate were tetradecanoic, hexadecanoic, hexadecenoic, octadecenoic, and C1,, cyclopropane fatty acids. The predominant fatty acids of strains NCS1, NCS2, and NCS3 grown without sodium oleate were dodecanoic, tetradecanoic, hexadecanoic, hexadecenoic, and octadecenoic acids. Stain NCS4 grown without sodium oleate contained considerably more C19 cyclopropane fatty acid than the other three strains. Cells grown in sodium oleate in all cases contained larger percentages of octadecenoic and C19 cyclopropane fatty acids than cells grown without sodium oleate. Cells Fatty acid composition of lipid fractions from L. bulgaricus NCSl. Results from gas chromatographic analyses of lipid fractions from cells of strains NCS1 grown with and without sodium oleate are presented in Table 4. The same fatty acids were observed in all fractions. The major differences appeared to be the presence of greater amounts of octadecenoic and C19, cyclopropane fatty acids along with lesser amounts of the saturated fatty acids in cells grown in sodium oleate than in cells grown in the control medium. More C1,, cyclopropane fatty acid was present in the polar fractions than in the neutral fraction. The cells grown in sodium oleate had more octadecenoic acid in the neutral than in the polar fraction; the reverse was true for cells grown in the control broth. Relationship of fatty acid composition to death resulting from freezing. The amount of death that resulted from freezing was closely associated with the cellular content of C,1 cyclopropane fatty acid. Figure 2 shows the percent death plotted against the cellular content of Car cyclopropane fatty acid for the lactobacillus cultures. The line had a negative regression that was significant at P < 0.001. Similar comparisons involving dodecanoic and tetradecanoic acids revealed positive regression lines that were significant at P < 0.001 and P < 0.005, respectively. Octadecanoic acid also had a positive regression line that was significant at P < 0.025. C1, cyclopropane fatty acid content exhibited the smallest standard deviation from the regression line of all the individual fatty acids. The regression coefficient of percent death on percent total saturated fatty acids was significant at P < 0.001. cyclopropane fatty acid content of L. bulgaricus. DISCUSSION Results from this and a previous study (25) suggested that sodium oleate was the active portion of Tween 80 responsible for producing cells that were stable to freezing in liquid nitrogen. The incorporation of [1-4C ]sodium oleate into the lipid fraction of cells of certain lactobacilli has been reported (17,21). The amount of labeled sodium oleate incorporated into the lipid fractions of L. bulgaricus NCS1 was similar to the percent of each fraction on a weight basis, which suggested that the incorporation was random and not selective for a specific lipid fraction. The total amount of cellular lipids and the ratio of polar to neutral lipid were similar to those of other lactobacilli (12,13). Since cyclopropane fatty acids are usually associated with phospholipids of bacteria (5,11) it was not surprising that the polar lipids of L. bulgaricus NCS1 contained more C,19 cyclopropane fatty acid than the neutral lipids. The phospholipid fraction thus appears to be important in protecting L. bulgaricus during freezing. The data indicate that the oleate was incorporated into the lipids intact or was converted to C19 cyclopropane fatty acid. Such a conversion of octadecenoic or cis-vaccenic acid to C19 cyclopropane fatty acid has been demonstrated in other lactobacilli (17,21 Generally, the major fatty acids of all strains were similar to those reported by Veerkamp (27) for L. bulgaricus. Alteration of the growth medium resulted in changes in the relative percentages of the individual fatty acids present in the cells. Similar effects of growth medium composition on the fatty acid content have been reported for other bacterial cells (14,27). The increases in octadecenoic or C19 cyclopropane fatty acid, accompanied by decreases in saturated fatty acids that were observed when L. bulgaricus cells were grown in the presence of sodium oleate, can be explained by metabolic control mechanisms involving these fatty acids (1,9,28). The lipids of gram-positive microorganisms, which are found predominately in the cell membrane (13), are important in maintaining membrane structure. The primary site of damage to certain cells during freezing is the cell membrane (19). Thus, the fatty acid composition might be involved in maintaining cell membrane integrity during freezing. Kodicek (16) suggested that cyclopropane fatty acids prevent close packing of lipids in cell membranes, making them more elastic and flexible during exposure to adverse environmental conditions. Jungkind and Wood (Abstr. Annu. Meet. Amer. Soc. Microbiol., p. 143, 1972) reported that strains of Streptococcus faecalis deficient in cyclopropane fatty acids were more sensitive to deoxycholate, NaCl, sodium lactate at pH 4.0, and incubation at 47 C than the parent strain that contained more of these acids. However, work on liposomes prepared from structural lipids of Escherichia coli suggested that the formation of cyclopropane acids from unsaturated fatty acids does not alter their physiochemical properties (8). The results from the present study to support the flexible membrane theory proposed by Kodicek (16) and the work of Jungkind and Wood (Abstr. Annu Meet. Amer. Soc. Microbiol., p. 143, 1972). A relationship has been shown between the amount of unsaturated and saturated fatty acids and fragility to the cell membranes of Mycoplasma laidlawii (23,24). Gier et al. (7) demonstrated that an increase in double bonds in the fatty acids of liposomes increased their permeability. Plant mitochondria that were sensitive to chilling had a higher content of saturated fatty acids than chill-resistant plant mitochondria (18). Chill-sensitive mitochondria were apparently injured due to an inflexibility of the membrane at low temperatures. A similar phenomenon may exist with regard to L. bulgaricus, because cell death appears to be related to a decrease in saturated fatty acid content. Both unsaturated and cyclopropane fatty (5). However, in the case of L. bulgaricus C1, cyclopropane fatty acid appears to be the one most closely related to the resistance of cells to freezing. The exact mechanism of protection is not known, but death is closely related to both the amount of saturated and cyclopropane fatty acids. Perhaps the cause of increased protection is due to a favorable balance of C19 cyclopropane and saturated fatty acids. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant ES-61 from the Division of Environmental Health Sciences.

v3-fos

2018-04-03T06:07:50.947Z

1974-04-01T00:00:00.000Z

45679513

s2

Stomach fermentation in East African Colobus monkeys in their natural state. The microbial fermentation in the stomachs of two monkeys, Colobus polykomos, collected in Kenya, was studied. The gas accumulated within the stomach contained H(2) but no CH(4). Volatile fatty acid concentrations were high, but accumulated acid prevented determination of the fermentation rate in untreated, incubated stomach contents. Upon addition of bicarbonate, a very rapid rate could be demonstrated. Some D- and L-lactate were in the stomach contents. Starchy seeds or fruits rather than leaves appeared to have been consumed. Microscopically, the most prominent microorganisms seen were large, very refringent cocci, possibly Sarcina ventriculi, and various smaller cocci and rods. The 28 cultured strains of bacteria included 14 Staphylococcus, 2 Streptococcus, 10 Propionibacterium, and 2 Peptostreptococcus. The culture count constituted 10 to 20% of the direct count. No protozoa or cellulolytic bacteria were found. An active microbial fermentation in the stomach of leaf-eating monkeys has been inferred (14) from the large amount of digesta in the stomach, from production of methane, and from the relatively high concentration of volatile fatty acids in the stomach contents (8). Measurements of fermentation in the langur monkey, Presbytis cristatus (3), and in Procolobus and Presbytis (14) indicate rates comparable to those reported for stomach contents of domestic (6) and wild ruminants (12). A brief field trip in Kenya in September 1969 afforded an opportunity to obtain two individuals of Colobus polykomos living on the north slope of Mt. Kenya and to culture the stomach contents and measure the rate of fermentation. MATERIALS AND METHODS Animals. Two male specimens were collected immediately adjacent to the Landrover containing the equipment used in the study. The first animal, no. 263, was in the Timao Forest on the north slope of Mt. Kenya at an elevation of about 3,200 m; the second, no. 264, was on the Burguret River on the lower edge of the timberline at an elevation of about 2,000 m. 'Present address: Institut fur Lebensmittelhygiene, Freie Universitat Berlin, 1 Berlin 33, West Germany. 2Present address: Veteringrische Anatomische Institut, 6300 Giessen, West Germany. ' Present address: Department of Animal Physiology, University of Nairobi, Nairobi, Kenya. Each was weighed and dissected as rapidly as possible. For monkey 263, samples of the gas in the stomach were removed 27 min after the animal was killed. For monkey 264 the gas was sampled at 21 min, and the pH was sampled after 26 min. The contents of the glandular portion of the stomach of 263 were almost white in color and were finely comminuted. The contents of the saccular portion were about as well comminuted but had a slightly green color, presumably due to ingested leaves. Leaves appeared to be a minor component of the diet. The stomach contents of 264 contained almost no green material. A number of large whole seeds (about 1 cm in diameter) were observed in 263. They were somewhat more abundant in the glandular than in the saccular portion of the stomach. Material from the saccular stomach of each animal was used for the culture and rate experiments. A sample of the contents from the saccular stomach of each animal was fixed with approximately 1 volume of acid-free Formalin. Direct microscopic counts of the bacteria in these samples were made with a Petroff-Hauser counting chamber and corrected for the dilution by the Formalin. Some of the contents of the glandular portion of the stomach of colobus 264 was Formalin fixed and later examined microscopically. Field equipment. Transportation by air and in the field necessitated miniaturization of equipment and materials. small cylinder (50 by 265 mm) provided with a needle valve outlet. The valve was threaded into a 15-mm opening at one end of the cylinder. To replenish the gas supply, the needle valve assembly was unscrewed and the cylinder was filled with powdered dry ice. The assembly was then screwed tightly into the cylinder, the valve was opened, and carbon dioxide was allowed to escape for 5 or 10 min. This washed out other gases. The CO2 was used untreated from the cylinder; facilities for scrubbing traces of oxygen were not feasible in the field. Small Pyrex culture tubes (10 by 75 mm) were provided with special butyl rubber stoppers (Jenkyns Rubber Co., London, England) (8 mm in diameter at the small end and 11 mm at the large end, 20 mm long). A small hole was drilled in the center of the upper surface, extending two-thirds through the length of the stopper to permit easier penetration by a syringe needle. Prior freezing in dry ice facilitated boring the hole with an ordinary drill press. The tubes were gassed out with carbon dioxide, stoppered, and sterilized before being taken into the field. Monoject 1-ml sterile disposable syringes, graduated in 0.01-ml divisions, fitted to 1-inch (about 2.5 cm), 20-gauge disposable needles (Becton Dickinson and Co.), were used for quantitative dilutions and for subcultures. In the latter case, the inoculum in only the dead space of the syringe and needle was transferred. Disposable 10-ml syringes were used for filling the tubes and for measuring out media. Media. Media used for initial cultivation included sheep rumen fluid cellulose broth, sheep rumen fluid without added carbohydrate, sheep rumen fluid cellulose agar (SF), colobus stomach fluid agar (CF), and colobus stomach fluid cellulose agar (CC). The methods of anaerobiosis were essentially as described earlier (10), except that the later modification using syringes and needles (11) was substituted for the earlier dilutions by pipettes and the gassing with glass capillaries. CF medium was prepared in the field. Some of the stomach contents were diluted with an equal volume of water, mixed, and filtered by squeezing the mixture through several layers of cheese cloth. The filtrate was used as two-thirds of the final medium. The remainder was a mixture of equal parts of mineral solutions A (0.6% NaCl, 0.3% KH2PO4, 0.15% (NH4)2SO4, 0.06% CaCl2, and 0.06% MgSO4) and B (0.3% K2HPO4). Ionagar (Colab laboratories, Inc., Glenwood, Ill.) at a 0.7% concentration was melted in the mineral mixture before addition of the stomach fluid. Resazurin (0.0001% final concentration) was included in all media. The medium, total volume of 18 ml, was placed in a 30-ml Pyrex Kjeldahl flask with a shortened neck, constricted to take the small stopper used on the culture tubes. After being autoclaved, the medium was cooled to 50 C, and the required amounts of 10% NaHCO, and 2.5% cysteine hydrochloride were injected and mixed to give final concentrations of 0.5 and 0.025%, respectively. The melted medium was tubed in 1.8-ml amounts and kept in a bucket of water at 50C until inoculated. The temperature of water baths was maintained by addition of boiling water as needed. CC medium was similarly prepared except that a pebble-milled suspension of 2% filter paper cellulose composed one-third of the medium, undiluted colobus stomach fluid composed one-third, and equal parts of mineral solutions A and B constituted the remainder. The sheep rumen fluid broth (one-third each water, sheep rumen fluid filtered through cotton, and mineral solution AB), sheep rumen fluid agar (same as broth but agar added), and SF (2% pebble-milled cellulose suspension substituted for the water) had been prepared in the culture tubes at Cambridge, England, by using rumen fluid from a sheep fed on grass. No additional substrate was added to the rumen fluid agar medium since previous experience with the rumen indicated that the colony count obtained without added substrate was about as high as with substrate and, though the colonies were smaller, longevity was greater, presumably because of decreased metabolite production. The bicarbonate and cysteine were added to the tube after the medium was melted and ready to be inoculated in the field. The colobus stomach contents were the consistency of a thick paste but were sufficiently liquid that, after mixing, a 1-ml sample could be sucked up into the calibrated portion of a 5-ml measuring pipette broken off squarely at the 5-ml calibration mark and provided with a rubber mouth tube. The sample was transferred to a tube containing sheep rumen fluid broth, the tube was gassed out, and the contents were thoroughly mixed. A 0.2-ml portion was then injected into a second rumen fluid dilution tube and further serially diluted through seven more tubes. From these, 0.1 ml was inoculated into each tube of the various culture series. After removing the inocula from the tubes of the original sheep rumen fluid dilution series, 0.2 ml of 2% cellulose suspension was injected into each tube to test for cellulose digestion that might occur in liquid but not in agar cultures. After inoculation, the tubes from the first monkey Were rolled in ice water, labeled, and incubated in the investigator's pocket. They were taken to the guest house overnight, where they were placed in a vacuum bottle containing water at 45 C. This had cooled by morning, when the temperature was adjusted to 39 C. The cultures from monkey 264 were placed in the vacuum bottle immediately after the agar had solidified, and that same day were transported back to the laboratory at Muguga, where all tubes were incubated at 39 C. After 8 days, the incubated tubes were examined and the colonies were counted in the higher dilutions showing growth. Individual colonies selected as representative of those observed were subcultured to a similar medium and also to rumen fluid glucose agar. In the higher dilutions containing fewer than 12 colonies, all colonies were subcultured. Subcultures were made at Muguga with the intent of shipping them immediately to California for further study, but permission to import was not obtained in time and it was necessary to take the cultures to New Zealand, where they were maintained until they could be sent to California. Between the initial subculture and the final characterization, a number of the strains were lost. Zero time rate measurement. A 40-g sample of the STOMACH FERMENTATION IN COLOBUS MONKEYS fresh stomach contents was placed anaerobically in a rubber-stoppered container and immersed in the water bath at 39 C. At 0, 15, 30, and 60 min for colobus 263 and at 0, 21, and 41 min for 264, a subsample of the incubated material was removed to a previously weighed plastic tube containing 1 ml of 10 N NaOH. The subsample was mixed with the alkali, and its weight was determined by difference. The alkaline samples were shipped to Davis by air freight and were analyzed immediately for volatile fatty acids. Nonvolatile acids in these samples were determined in 1971 after adding water to make up that lost by evaporation during storage. Gas analyses. A sample of the gas in the stomach was taken by syringe before the stomach wall was opened. The short sampling needle was replaced with a 20-gauge, 6-in (about 15 cm) needle, and the gas was transferred to the base of a dry culture tube (16 by 150 mm), drawn out to a capillary in its central portion. Some of the gas sample was used to flush out the base of the tube, and the narrow portion was then quickly sealed with a propane torch to enclose the sample in an all-glass container. The'samples were sent to Davis and analyzed with a Perkin-Elmer model 154B gas chromatograph, using a silica gel column and N2 as carrier gas. Analysis of fermentation products and substrates. The pH of the stomach contents was tested with paper having a range of 5.5 to 7.0. Volatile fatty acids in the stomach contents and in the products of the metabolism of pure cultures were determined on a 700 F and M gas chromatograph with a flame ionization detector and a 6-ft (about 1.9 m) column filled with FFAP on Chromosorb W. Carrier gas was helium at a flow rate of 40 ml/min. Column temperature was 125 C, injection port was 210 C, and detector was 290 C. Membrane-filtered (0.22 uim pore size; Millipore Corp.) acidified supernatant fluid was injected directly into the column, or, in analyses of pure cultures, the volatile fatty acids were first distilled at low temperature in a closed vacuum system with the receiving tube dipped in ice water. Qualitative examination for nonvolatile acids was performed by spotting acidified stomach contents on a thin-layer chromatographic plate of ECTEOLA cellulose 300 (Macherey, Nagel and Co.), separating the acids by development with ethanol-water-NH4OH (16:3: 1) and detecting with a spray of 0.04% bromothymol blue adjusted to pH 8.0. For quantitative measurements, samples were first made alkaline and evaporated to dryness; they were then acidified and the volatile fatty acids were separated by vacuum distillation. The residue was extracted with three 5-ml volumes of freshly distilled anhydrous peroxide-free ether. Water (0.2 ml) was added, and the acids were titrated. The ether was evaporated off at room temperature, and water was added to a total volume of 1 ml. L(+)-Lactate in this material was determined with the Lactostat Kit (Sigma Chemical Co.). This involved enzymatic oxidation to pyruvate and reduced nicotinamide adenine dinucleotide, the latter being determined spectrophotometrically. D(-)-Lactate also was determined from enzymatic nicotinamide adenine dinucleotide reduction by using D(-)-lactic dehydrogenase (Boeh-ringer Co., Mannheim). In both reactions, the pyruvate formed was removed by a pyruvate-glutamate transaminase reaction Total nitrogen was measured by the Kjeldahl method. The anthrone method was used for carbohydrates. To determine the starch content of the Formalinfixed stomach contents, a 75-mg sample was dried to constant weight and suspended in 25 ml of water; 1 ml of human saliva was added, and a 10-ml sample was immediately cooled in an ice bath and centrifuged at 4 C. The remainder was incubated for 24 h at 37 C, and a 10-ml sample was similarly centrifuged. The increase in anthrone values of each supernatant after saliva treatment was used as a rough measure of the starch content. Total carbohydrate was estimated by taking 0.1 ml of the above suspension and analyzing it by the anthrone method. For analyses of the fermentation products of the pure cultures, the strain was inoculated into either peptone beef extract broth (PB) or, for some requiring additional nutrients, into dilute rumen fluid broth. PB contained 4.5 g of peptone (Difco), 2.25 g of beef extract, 0.45 ml of 0.1% resazurin, 210 ml of deionized water, 75 ml of mineral solution A, 75 ml of mineral solution B, 90 mg of cysteine hydrochloride, 11.25 ml of 4% glucose solution, and 33.75 ml of 10% NaHCOs solution. The rumen fluid medium differed in containing only 0.135 g of peptone and 0.135 g of beef extract, with 45 ml of rumen fluid clarified by centrifugation replacing 45 ml of water. To assure identical CO2 content in all tubes, the medium (except for the glucose and bicarbonate) was prepared in a round-bottom, 1-liter flask, equilibrated at room temperature with 100% CO2, sealed, and sterilized. The sterilized medium was cooled to room temperature and then opened, with a gassing needle to exclude air from the flask but without bubbling the CO2 through the medium. The sterile glucose and bicarbonate solutions were then added aseptically and anaerobically and mixed, and 9 ml of the medium was transferred anaerobically tp the sterile culture tubes, each containing 1 ml of water. Air was continuously excluded also from the culture tube without bubbling the contents, and the tubes were closed with a recessed butyl rubber stopper. They were inoculated with 0.2 ml of culture, and some were refrigerated as a control. The same media without glucose were inoculated and incubated as a control for fermentation products formed from substrates other than glucose. After completion of growth at 37 C, the cultures were allowed to come to room temperature and the gas produced was measured. A 1-inch, 21-guage needle, attached to a 20-ml syringe well lubricated with water, was inserted through the stopper, and the contents of tube and syringe were equilibrated by vigorous shaking. The amount of excess gas in the syringe was recorded. Without removing the first syringe, 1 ml of normal HCl was injected and, after equilibration, the total gas volume was again read. The second volume minus the first gave a measure of the residual bicarbonate in the tube. The difference between the second volumes in the incubated and in the refrigerated tubes gave the volume of gas pro-VOL. 27, 1974 duced in metabolism, aside from that released from bicarbonate. The difference in residual bicarbonate gave a measure of the amount of acid produced during metabolism. The differences in gas volumes for duplicate cultures differed usually by less than 2%. One milliliter of 2 N NaOH was then injected, still without removing the initial syringe, and the CO2 was absorbed. Ten milliliters of N2 was injected to maintain a gas pressure above atmospheric. This third volume was read, the gas in the syringe was injected back into the tube, and the syringe was withdrawn. The difference between the gas absorbed in the incubated and refrigerated cultures gave the amount of CO2 produced or used. The gas in the tube was analyzed for H2 by injecting 0.5 ml into a Perkin-Elmer 154B gas chromatograph with a silica gel column and N2 as carrier gas. Peak heights were compared with a standard. Fermentation rate measurements. A volume of mineral solution containing 0.5% NaHCO, was added to an equal volume of stomach contents in each of three wide-mouth bottles closed with a rubber stopper perforated by a 2-inch (about 5 cm) needle attached to a water-lubricated 10-ml glass syringe (16). An initial interval was allowed for temperature equilibration to 39 C and to check the uniformity in the rate of gas production in the three bottles. Strong acid was then added to one of the bottles as a control to release all CO2 from the bicarbonate. Differences in residual bicarbonate gave a measure of the difference in acid production. The total gas released during the fermentation minus that due to acid production gave a measure of the amount of gas produced in metabolism. Before each reading, the contents of each bottle were vigorously shaken by hand to equilibrate the CO, between liquid and gas phases. The amounts of gas evolved were corrected to standard conditions. A temperature of 39 C and vapor pressure of 52 mm of Hg were assumed, though the actual temperature and vapor pressure were somewhat lower than this because the measuring syringe was not immersed in the water bath. Barometric pressures of 522 and 564 mm of Hg (=6.95 x 10' and 7.5 x 10' N/in) were assumed for the experiments with colobus 263 and 264, respectively. The pressure increase due to the weight of the syringe barrel, 10 g/cm2 (=9.806 x 102 N/M2), amounted to less than 1.5% of the total pressure and was neglected. All of these errors made the reported rates of gas production slightly less than the actual. Determination of guanosine-cytidine percentages. Strains identified as Staphylococcus were treated with lysostaphin (Schwartz/Mann, Orangeburg, N.Y.) according to Klesius and Schuhardt (13), and the base composition of the recovered deoxyribonucleic acid was estimated from its buoyant density in a cesium chloride gradient by using a Spinco model E analytical centrifuge. Tests on Staphylococcus aureus. For production of enterotoxin, strains were grown in sac cultures according to the method of Donnelly et al. (7). The amount of enterotoxin formed was measured according to the microslide method of Untermann (17). Stan-dard disks were used to measure antibiotic sensitivity. For arsenic and cadmium sensitivity measurements, disks were impregnanted with 250 gg of Na2HAsO4 7H20 or 3.1 ug of Cd(N02)2 4H20. RESULTS Measurements in the field and many of those from the laboratory are shown in Table 1. The results of analyses of zero time rate samples are shown in Table 2. These were from gas chromatographic analyses completed in the fall of 1969 just after the materials were shipped to Davis. The zero time rate experiments were started 41 min after colobus 263 was sacrificed and 35 min after colobus 264 was sacrificed. The Formalin-fixed stomach contents, analyzed in 1971, contained roughly the same kinds and quantities of fermentation products. The Formalin had been specially purified for fixation of tissues and was free of acids. In 1971, the zero time rate samples were examined also for nonvolatile acids. The thinlayer chromatography plates did not show any succinic acid but did show a little lactic acid. For neither one did the relative size of the spots indicate that their concentration increased during the incubation period. The amounts of lactic acid are shown in Table 2, including the results of analyses for D-and L-lactate on the Formalin-fixed stomach contents. Fermentation rate. In the experiment with stomach material from colobus 263, three bottles were incubated, each containing 42 ml of colobus stomach contents (saccular portion) mixed with 42 ml of balanced salt solution containing 0.5% NaHCO,. The samples were incubated at 39 C, and the gas production was measured to determine whether the rates in the three vessels were comparable. After 15 min of incubation, excess acid was added to one vessel, releasing 4.3 ml (corrected) of gas from bicarbonate. The other vessels, incubated an additional 10 min before acid was added, also showed only 4.3 ml of gas liberated by the acid. The amount of bicarbonate remaining after equilibration was apparently insufficient, and the experiment did not measure the fermentation acids formed. The rates of gas production in the three vessels were 31, 27, and 25 ,umol per h per g of fresh stomach contents, respectively. In the run the next morning with colobus 264, only 32 g of stomach contents was used, and the bicarbonate solution was increased to 50 ml. Figure 1 shows the gas evolution in the three vessels and also the amount of residual bicarbonate in each when acid was added. The rates of gas production in bottles 1, 2, and 3 were 148, 194, and 195 Mimol per g per h, respectively. Comparison of bottles 2 and 3 between 17 and 23 min (Fig. 1) shows a difference of 7.1 ml (corrected) of CO2 released from the residual bicarbonate, indicating that 316 ,gmol of the gas evolved during this part of the fermentation was CO, released from bicarbonate by the fermentation acids. The total gas produced and released by acid in bottle 2 during this period was 408 umol, giving 92 Mmol as the amount of CO2 and H2 produced in the fermentation and 316 ,mol as the fermentation acids. In calibration experiments done previously in California with bovine rumen contents plus mineral solution AB containing 0.5% NaHCO,, incubated in an atmosphere of CO2 (giving a pH of about 6.7), the amount of CO2 released by added lactic acid was equivalent to only about 70% of the amount expected if eq of acid liberates 1 mol of CO2. Correction for a similar lack of stoichiometry in the colobus 264 experiments gave a ratio of fermentation acid to gas of about 5. For bottles 1 and 3 between 17 and 22 min, 132 ,mol of CO2 was released by fermentation acids, and 118 Amol of CO2 and H2 was produced in metabolism. With the 0.7 correction, the ratio of acid to gas was 1.6. The low ratio can be explained in part by the longer incubation of sample 1, with consequent greater depletion of bicarbonate. The experimental error was fairly large in these determinations because of the short incubation time. Nature of the bacteria. The Formalin-fixed stomach contents were examined at Muguga with an oil immersion phase microscope. The microscopic appearances of the two animals were remarkably similar. Large cocci arranged in diplo and tetrad forms were morphologically the most distinctive bacteria observed. Some of them were very refringent, bright in appearance under dark-phase illumination, and regularly arranged in twos or tetrads. A few spirochetes were seen. Photomicrographs of the Formalinfixed material gave the appearance shown in Fig. 2. In colobus 264, the large refringent cocci were relatively more abundant in the contents from the glandular region of the stomach as compared with the forestomach material. The former was not collected from colobus 263. Culture counts for colobus 263 on the CF series and on the sheep rumen fluid series showed 2.2 x 109 and 7.7 x 109 colony-forming units per ml of fresh forestomach contents, respectively. For colobus 264, the counts were the same for both of these media, namely 2.6 x 10g. Direct counts on the Formalin-fixed material were 3.8 x 1010 and 2.6 x 10'0/ml for colobus 263 and 264, respectively. None of the culture series containing cellulose showed any cellulose digestion even after 7 weeks of incubation. Two large, non-cellulolytic colonies in tubes 5 and 6 of the CC medium were subcultured as strains 1-6cc-1 and 1-7cc-1. In this strain designation, the first number indicates the first, 263, or second, 264, monkey. The second number is the dilution tube from which the colony was picked, with the letters indicating the medium (cf for colobus fluid agar, cc for colobus fluid cellulose agar, and sf for sheep rumen fluid agar). The third number is that of the colony picked, the letter following being used to designate the strain when more than one was isolated from that colony. Ten colonies were subcultured from the eighth dilution tube of the CF agar series inoculated from animal 263, and five were subcultured from the ninth dilution. Ten colonies were subcultured from the eighth dilution tube of the CF series inoculated from 264, five were subcultured from the eighth dilution of the sheep rumen fluid agar series, and nine were subcultured from the ninth dilution. Three first subcultures and some subsequent subcultures failed to grow even though some colobus fluid was included in the medium. Of the remaining picked colonies, 27 survived further vicissitudes of shipment and various mishaps. From one of them two different bacteria were isolated, giving a total of 28 strains that were studied. Sixteen of the 28 strains characterized were euryoxic, of which 14 formed catalase. These 14 were gram-positive cocci, in single, diplo, tetrad, or irregular chain arrangement, and fermented glucose without producing visible gas bubbles. The diameter of the cells varied from 0.6 to 1.2 ,m, and in some strains was quite variable. Nine of the strains formed white colonies, of which four strains liquefied gelatin; four of them were light yellow and did not liquefy gelatin. One strain was golden yellow and fermented mannitol, whereas the others did not. It reduced nitrate but did not liquefy gelatin. The golden strain (2-8sf-2) was identified as S. aureus. It was identical to S. aureus ATCC 14458 in morphology and in production of coagulase, proteinase, lipase, nuclease, and phosphatase. ATCC 14458 gave only delta-hemolysis on sheep, cow, rabbit, and human blood, whereas strain 2-8sf-2 showed a hemolysin for all bloods tested and was beta-hemolytic on sheep and cow blood. Strain 2-8sf-2 also differed in being sensitive to arsenic and cadmium, streptomycin, tetracycline, and penicillin. It was negative for production of enterotoxins A, B, C, and D ( (4). The other 13 euryoxic catalase-positive strains (Table 4) also showed the characteristics of the genus Staphylococcus and were presumably S. epidermidis (1). For several representative strains the guanosine-cytidine percentages were determined (Table 5). Lactic acid was the chief fermentation product of the three tested strains of staphylococcus grown anaerobically on glucose ( Table 6). The recovered lactate accounted for 75 to 88% of the glucose provided. The lactate was chiefly the L-form for two of the strains. The third strain formed almost equal amounts of L-and D-lac-tate. The acetate in the experimental culture was no greater than in the uninoculated control. The calculated yield of cells was low for a homolactic fermentation, only 11.2, 13.2, and 10.6 gg of cells per Amol of glucose for the three cultures, respectively. The yield of cells was estimated from the nitrogen content of the sediment of the culture, because it was necessary to add CaCO, to obtain good growth. In an experiment without CaCO,, the nitrogen content of the cells of strain 2-9sf-4 was 12.6% of the dry weight. They were nonmotile and without capsules. They produced large, circular, entire, thin, lenticular, deep colonies and opaque, smooth, white colonies on the surface of the agar in the roll tubes. Three tested strains (1-8cf-2b, 1-8cf-10, and 2-8cf-4) were catalase positive and produced acetic and propionic acids in the ratios expected for Propionibacterium. The production of H2 varied (Table 7). A slight amount of H2 was produced by all three strains when the medium contained 0.5% peptone and 0.3% beef extract, and only in strain 1-8cf-10 was the H2 production increased by glucose. One strain produced a slight amount of H2 from rumen fluid. All three strains grew on the glucose-free peptone, and the amount of fermentation products was not greatly increased by addition of glucose ( Table 8). The eight strains were assigned to the genus Propionibacterium. The other four anaerobic strains were not as easy to classify. Cells of strain 1-8cf-9 ranged in shape from cocci to rods, 0.5 Am in diameter, single, in V's or close clusters. The Gram reaction was not clearly negative or positive. The strain produced 9 Amol of acetic acid per ml when grown on PB, and this amount was not increased if glucose was added. No propionic acid was formed on the PB medium, but if glucose was added, 1 gmol of propionic acid per ml was formed. The strain did not produce H2 at any time and is probably closely related to Propionibacterium. Strain 2-8sf-1 contained gram-positive rods to cocci, single, in pairs or chains, 0.75 to 1.25 Am in diameter and 1.25 to 2.0 Am long. They were nonmotile and without capsules, and formed acetic and propionic acids in the ratio expected for Propionibacterium, but they also produced a great deal of H2 (Table 7). In its fermentation products, the strain appeared to be intermediate between Propionibacterium and Veillonella. A slight amount of H2 was formed on PB or rumen fluid medium alone, but addition of glucose caused copious H2 production. There was very little growth on rumen fluid medium alone but a fair amount with glucose added. Even with added glucose, the optical density on rumen fluid medium was not as great as that on PB, which was about half of that on PB plus glucose ( Table 7). Traces of n-butyric and isovaleric acids were formed, the amounts being slightly increased by glucose. Strain 1-9sf-2 was non-saccharoclastic and formed some acetate and H2 from the PB medium without glucose. It grew very poorly on the few media tested. Morphologically it consisted of chains of cocci and is probably referable to the genus Peptostreptococcus. No propionic or butyric acid was formed. Strain 2-9sf-1 resembled 1-9sf-2 in morphology and culture characteristics. DISCUSSION The results from the zero time rate experiments demonstrate that within 30 min after death of the animal, the fermentation was inhibited by the acids formed, in agreement with the observations of Kuhn (14). Rapid acid production was indicated by the in vitro fermentation rate experiments. With colobus 263, the bicarbonate added was insufficient to bring the pH into the range (about pH 7.0) at which the acids produced could be measured by release of CO2 from bicarbonate. In this experiment, 2,520 Amol of NaHCO. (60 ,omol/ml) were added. Addition of excess acid at 34, 44, and 45 min of incubation of the three a Optical density (OD) was measured at a wave length of 600 nm in a cuvette with a 1-cm light path. The acid production in the live monkey stomachs must have been considerably greater than in ruminants collected in nature and similarly studied (12). In the ruminant studies, only 30 Mimol of NaHCO, per ml of rumen contents was sufficient to maintain an excess of bicarbonate during the in vitro fermentation rate measurements made at about the same time after death of the animal. With colobus 264, 99 ,gmol of NaHCO, per ml of stomach contents was added, and in this case 48, 714, and 536 Mmol of gas were released by acid added after 17, 23, and 24 min of incubation, respectively, as compared with the 3,168 ,gmol total of added bicarbonate. With colobus 264, the total gas released, both during the run and by acid added later, amounted to 2,076, 2,634, and 2,813 ,umol for the three samples, respectively. The more rapid development of acidity as compared with the rumen could be due to a greater availability of substrate in the colobus, to poorer buffering, or to poorer absorption. The stomach contents appeared to be chiefly finely comminuted, starch-like white material and gave an intense black color when tested with iodine, though the percentage digestible by saliva was less than 5% ( Table 1). The fermentation gas production rate for the first animal, 28 gmol per h per g (wet weight), is of the same order of magnitude as the values of 38 and 12 reported by Kuhn (14) for Presbytis cristatus and Procolobus badius, respectively. Values of 63 to 79 umol per h per g reported by Bauchop and Martucci (3) for Presbytis cristatus are considerably greater but are less than the average rate of 179 ,umol per h per g for colobus 264. This high rate represents the potential for a very rapid fermentation when sufficient bicarbonate is added to maintain a favorable pH. It is doubtful that the high rate for colobus 264 obtained in the animal. More likely, after feeding, acid is produced more rapidly than it can be absorbed, and the resulting acidity slows the fermentation. The slower rate observed with colobus 263 is unexplained, but may be due to a longer interval between the last feeding and the time of sampling. It seems unlikely after death that mixing of the acidic contents of the glandular stomach contents with the forestomach contents could account for the high acidity of the latter, since the contents of both compartments were relatively dry. The percentage of dry matter in the saccular stomach contents of colobus 264 (Table 1) was much higher (31.7%) than in colobus 263 (18.7%), and the percentage nitrogen was much lower. Colobus 264, collected at 8:32 a.m., may have completed a morning feed on dried seeds and not yet consumed water. The ratios of the concentrations of the various fermentation acids in the zero time rate samples and in the Formalin-preserved material ( Table 2) are similar to those typical of rumen samples. For colobus 264, the total concentrations of acids is about as high as the highest values encountered in the rumen. There is more lactate in the colobus stomach than is usually found in forage-fed ruminants but comparable to those on a high concentrate ration (2). The absence of methane from the gas in the colobus stomach contrasts with its occurrence in the langur (3) and in the ruminant. The stomach fermentation of the colobus differs also from that in a marsupial, the quokka (Setonix brachyura) (Moir and Hungate, unpublished data), which formed significant quantities of both hydrogen and methane. Another difference in the colobus fermentation from that in ruminants is the absence of cellulolytic bacteria. No bacterial colonies surrounded by zones cleared of cellulose were observed in any of the cellulose agar cultures containing rumen fluid or colobus fluid, and none of the liquid cultures showed any disappearance of cellulose. These negative results might be explained in lower dilutions as due to too great an acidity introduced with the inoculum, but this could not explain the negative results in the higher dilutions. The stomach contents seemed to contain very little plant fibrous material, suggesting that "leaf eating" may not apply to Colobus polykomos. Acidity may be a factor preventing the development of both methanogenic and cellulolytic bacteria in the colobus stomach. Acidity may also explain the absence of protozoa, though Kuhn (14) postulated that it was the consistency of the contents which prevented protozoal growth. Purser and Moir (15) found that the rumen protozoa could not survive continuous exposure to acidities much below pH 6. Another difference from the forage-fed ruminant is the greater number of euryoxic cultures as compared with anaerobes. Inability to absorb traces of 02 from the CO2 used in the field experiments might be a factor in the increased proportion of aerobes and in the low ratio of culture count to direct count. But the absolute number of cultured aerobes per milliliter is much higher than for the rumen. The culture counts were 20 and 10% of the direct counts for the two monkeys, respectively. This is a slightly lower ratio than is obtained in careful culture experiments on rumen contents from forage-fed cattle, but is of the same order of magnitude. A higher ratio of culture count to direct count is usually obtained for ruminants on high concentrate rations. Although propionibacteria are occasionally abundant in the rumen (9), they are not usually a prominent element, nor are staphylococci commonly found. The morphology of the cultured bacteria is consistent with the morphology of some of those seen by direct microscope examination. The pure culture strain 1-8cf-4, a staphylococcus, showed tetrads of cocci very similar to the tetrads of smaller cocci in Fig. 2. None of the cultured strains resembled the very large refringent cocci. The appearance of these cells is similar to that of Sarcina ventriculi, and, in view of their greater abundance in the contents of the more acidic glandular stomach contents, it seems possible that this species is a normal inhabitant of the colobus stomach. The observed high refringence might be due to the cellulose produced by this species (5). It seemed to be more marked as the cells increased in size. The relative abundance of staphylococci in the colobus might be the result of grooming with the teeth for fleas and other external parasites, a process which could conceivably provide a continuous inoculum, proliferating further under the conditions in the stomach. Thorough examination failed to demonstrate any enterotoxin production by the isolated Staphylococcus aureus (Table 3). This suggests that the staphylococcus strains in the colobus stomach may have been selected for host digestive compatibility and may be a normal component of the stomach microflora, maintaining themselves without ingested inocula. The concentration of fermentation products affords some index to their rate of absorption, since absorption of volatile fatty acids is a function of the concentration gradient between stomach contents and blood. The concentration of volatile fatty acids in the stomach of colobus 263 ranged between 107 and 125 ,mol per ml of stomach contents and between 212 and 434 for colobus 264 ( Table 2). The values obtained from analyses of the Formalin-fixed material are roughly the same, if the dilution by the Formalin is taken into account.

v3-fos

2020-12-10T09:04:22.725Z

1974-08-01T00:00:00.000Z

237234471

s2

Effect of Potassium Sorbate on Salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in Cooked, Uncured Sausage Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate. Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate. Sorbic acid and its salts are used in many commercially prepared foods for preventing spoilage. Sorbic acid is selective in its antimicrobial activity. For example, it is a very effective inhibitor of yeasts in cucumber fermentations and yet permits the normal development of lactic acid-producing bacteria, except in those cases of high sorbic acid levels combined with a high initial brine content (3). Sorbic acid has been added to culture media for the selective isolation of catalase-negative lactobacilli and clostridia (5,7). All strains of Clostridium botulinum types A and B tested by York and Vaughn (8) were found to grow within 7 days at 35 C in beef liver infusion containing 3.0% sorbic acid. Hansen and Appelman (6) reported that sorbic acid neither inhibited nor stimulated the growth of C. botulinum in culture media. The observations that sorbic acid is selective in its antimicrobial activity and lack of inhibition for C. botulinum form the basis for a very restrictive use of sorbic acid and its salts in meat products. The only approved use consists of dipping the casings-for stuffed dry sausage. This application inhibits mold growth on the surface of the sausages during the long period they are held in drying rooms. Sorbate cannot be added to the meat portion of pizza pies even though it is permitted in the cheese and crust. Spoilage by molds results in significant loss of meat products at the retail and consumer levels. Addition of sorbates to the meat would reduce this loss of food. This study was designed to determine the effect of potassium sorbate on C. botulinum in cooked sausage in the event the product is temperature abused. Also, the effect of potassium sorbate on the growth of salmonellae, Staphylococcus aureus, and C. perfringens was examined. The methods of inoculating the product represent recontamination on the surface of the sausages after cooking (salmonellae and S. aureus) and spores surviving the cooking process (C. botulinum and C. perfringens). MATERIALS AND METHODS Meat. Cooked, skinless sausage links were used as the test system. The raw meat formula consisted of a mixture of beef and pork (87.95%), water (8.8%), sodium chloride (1.75%), and sugar and spices (1.5%). The fat content of the uncooked links was 43%. One lot of sausages was prepared in which 0.1% potassium sorbate (wt/wt; Mallinckrodt Chemical Works, Lodi, N.J.) was added to the ground meat formula prior to cooking. The links were heated to an internal temperature of 71 C and then frozen until needed. The cooked sausages had a pH of 6.2. The average weight of the links were 20 g each. Cultures. Five salmonellae cultures (Salmonella anatum, S. infantis, S. senftenberg, S. choleraesuis, and S. newport) were grown at 37 C in brain heart infusion broth (BHI; Difco). After incubation for 18 h, 262 the five strains were pooled for inoculation of the sausage. Five strains of S. aureus (S-6, 196E, 361, FDA 315, and ATCC 6538) were grown in BHI broth at 37 C and pooled after 18 h. Four of these strains are known to produce one or more of the known enterotoxins. Spores of three strains of C. perfringens (NCTC 8239, FDI, and ATCC 3624) were prepared as described by Duncan and Strong (4). The spore crops were harvested by centrifugation, washed several times in sterile distilled water, then resuspended in phosphate buffer (pH 7.0) and pooled to provide equal levels of each of the three strains for inoculation. Spores of five strains of type A (77A, 62A, 33A, 12885A, and 36A) and five strains of type B (9B, 40B, 41B, 51B, and 53B) C. botulinum were prepared as previously described (2). A pooled mixture of the 10 spore crops was used for inoculation of the sausage. Inoculation of meat. Our goal was to achieve an inoculum level of 1,000 C. botulinum and 1,000 C. perfringens spores per link and 1,000 salmonellae and 1,000 S. aureus per g of product. Sausages were inoculated on the outer surface with salmonellae by immersing the sausages in a dilute aqueous suspension of the pooled salmonellae. The sausages were removed from the suspension and allowed to drain. Each sausage link retained approximately 0.1 ml of the suspension and yielded an initial inoculum level of 32 to 38 per g or 640 to 760 per sausage link. The same method of inoculation for S. aureus yielded an initial level of less than 30 per g or less than 600 per sausage link. This inoculum level was below the limit of our method for enumerating S. aureus. The mixed spore suspension of C. botulinum was heat shocked at 80 C for 15 min and 0.1 ml was injected into the center of the sausage links. This yielded an inoculum level of more than 7,200 per g or 140,000 per sausage link, which was more than anticipated. The mixed spore suspension of C. perfringens was heat shocked at 70 C for 20 min before injection. The inoculum level was 320 per g or 6,400 per sausage link. The above methods were followed for product with and without potassium sorbate. Separate inoculum suspensions of salmonellae and S. aureus were used to dip sausages with and without sorbate. Storage and sampling of product. Samples representing each variable were packed separately in boxes (10 links per box) giving a net weight of about 200 g. Uninoculated control product was included for comparison. All product was stored at 27 C. Three boxes of each variable were removed for analysis at predetermined time intervals. A 90-g sample was removed from each box and blended in a Waring blender with 90 ml of sterile phosphate buffer (0.003 M, pH 7.0) for 1 min. After making appropriate dilutions, viable counts were determined. Total aerobic counts of uninoculated product were made by using standard plate count agar (Difco) with incubation at 27 C for 2 days. Salmonellae were enumerated by spreading onto brilliant green sulfa agar (Difco). Salmonellalike colonies were counted after 24 h at 37 C. Representative colonies were transferred into lysine iron agar slants (Difco) and examined for typical reac-tions. Samples inoculated with S. aureus were plated onto Baird-Parker agar (Difco) and examined after 24 h at 37 C. Samples inoculated with C. perfringens were plated into SFP agar (Difco), overlayed, and incubated in anaerobic jars at 37 C for 24 h. Counts were made and typical colonies were confirmed by transferring into nitrate-motility medium (1). Samples inoculated with C. botulinum were analyzed by a three-tube, most-probable-number technique using peptone colloid (Difco) modified by the addition of 0.1% dextrose, 0.05% ferrous sulfate, and 0.03% sodium thiosulfate. All tubes showing blackening after day 7 at 37 C were assumed to contain C. botulinum. Black tubes from the highest dilutions were selected at random and were confirmed by mouse test to contain botulinal toxin. Sausages inoculated with C. botulinum were also tested for toxin by centrifuging a portion of the 1: 1 suspension of the blended sample. Two white mice were injected with 0.5 ml of the supernatant fluid. A third mouse was injected with 0.5 ml of supernatant which had been boiled for 15 min. Death of the mice receiving the unheated extract and survival of the mouse injected with the boiled extract coupled with the C. botulinum viable counts were considered evidence for the presence of botulinal toxin. RESULTS AND DISCUSSION Potassium sorbate delayed the growth of the normal bacterial spoilage flora in the uninoculated product during the first day, after which growth was rapid ( Table 1). Growth of the salmonellae was markedy retarded by potassium sorbate. This agrees with a report (5) that sorbic acid inhibited salmonellae in culture media. Growth of S. aureus was inhibited during the first day, after which growth was rapid. C. perfringens declined to below detectable levels in all samples within the first day, independent of the presence or absence of potassium sorbate. Growth of C. botulinum was slower in the product containing potassium sorbate. Also, there was a delay in the development of botulinal toxin. Botulinal toxin was detected in 4 days in product without sorbate, but not until after 10 days in the product with sorbate. The product inoculated with C. botulinum had pH values of 6.4 and 7.1 after 4 days in samples with and without sorbate, respectively. A pH of 6.4 is sufficiently high to exclude the possibility that pH was a factor in the inhibition of botulinal growth and toxin production. These data demonstrate that the addition of 0.1% potassium sorbate to retard mold spoilage does not increase the public health hazard of cooked pork sausage in the event that it becomes temperature abused. To the contrary, the

v3-fos

2018-04-03T05:52:56.031Z

1974-04-01T00:00:00.000Z

23800679

s2

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 1010 to 7.64 x 1010 per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10-9) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media Numerous studies on the intestinal microflora of the domestic fowl, Gallus domesticus, have been made (2,3,5,23,26,30,34). In many of these investigations, selective plate media and conventional anaerobic jar methods have been used in an analysis of specific bacterial groups, namely, coliforms, streptococci, lactobacilli, bacteroides, and clostridia. At best only these groups of bacteria have been identified, and unless very good anaerobic techniques were used, some of the predominating anaerobic species have been overlooked. Others, however, have developed nonselective media for the isolation of rumen anaerobic bacteria (9,12). With these media (containing low levels of energy sources) in conjunction with strict anaerobic techniques, numerous bacterial types are permitted to grow. Relatively few studies on poultry intestinal microflora have been made using such media and methods. Recent work by Barnes et al. (3,5) on the isolation of anaerobes from the chicken cecum has indicated that 20 to 678 30% of the direct microscope count can be cultured by using the Hungate technique and a nonselective medium such as medium 10 (medium without rumen fluid devised for rumen anaerobes by Caldwell and Bryant [12]) supplemented with liver and chicken fecal extracts. However, data on comparison of other roll-tube media (e.g., rumen fluid-containing media used for culturing the predominant ruminal species) or the effect of various medium constituents on the enumeration and isolation of chicken cecal bacteria are not available. Experiments in this laboratory indicate that a rumen fluid medium can be used to isolate a large percentage of the total bacteria from chicken cecal digesta. In addition, our present studies compare the cultural characteristics and isolation efficiency of other nonselective rolltube media and furnish information on the presumptive identification and relative distribution of the predominant anaerobic bacteria colonizing the cecum of the chicken. MATERIALS AND METHODS Animals and diet. In experiments designed to determine colony counts, recovery of anaerobes, and effects of medium components in different nonselective media, cecal samples were obtained from 5-weekold cockerels (White Cornish cockerel x White Rock hen) supplied by a commercial broiler facility. In all other experiments on the cecal microflora including isolation and identification of bacterial strains, cecal samples were obtained from 5-week-old, "laboratoryreared" birds described below. One-day-old cockerels (same breed as above) were housed in temperature-controlled rooms (12-h light cycle and slight negative pressure) on floors covered with autoclaved pine shavings. An initial brooding temperature of 35 C was maintained for young birds and reduced in weekly increments to a constant temperature of 25 C at 3 weeks of age. Throughout the growth period, a commercial broiler grower ration and water were provided without restriction. The diet was supplemented with a coccidiostat continuously and with antibiotic (50 mg of bacitracin per kg of feed) from day 1 to 4 weeks of age. After 4 weeks, antibioticfree ration of the same composition replaced the medicated ration. Anaerobic culture techniques. Anaerobic techniques employed were similar to those described by Hungate (20,21) for rumen bacteria with modifications of Bryant (6,7). Strict anaerobic techniques were maintained throughout all procedures involving the dilution of cecal samples and preparation and inoculation of media. Sampling procedure. Five-week-old cockerels were taken at midday and sacrificed by CO2 asphyxiation. Immediately, cecal samples (2 to 4 g, wet weight) from both ceca of a single animal were placed in a sterile, stoppered tube and flushed with oxygen-free CO2. Cecal contents were weighed and processed through serial 10-fold dilutions in tubes of the anaerobic mineral solution of Bryant and Burkey (7). Fractions (0.2 and 1.0 ml) of a dilution (up to 10-9) were added to prereduced and melted agar tubes of the various media maintained at 48 C. Within 10 min after inoculation, roll tubes were made by rapidly rotating tubes in a spinner (Bellco Glass, Inc.) and simultaneously cooling with cold water. Solidified agar roll tubes were incubated up to 14 days at 37 C. Roil-tube media and media preparation. The compositions of different nonselective roll-tube media compared in this study are given in Table 1. Medium 98-5 is an improved rumen fluid medium for culturing ruminal bacteria and has been described by Bryant and Robinson (9). Modified 98-5 (M98-5) medium is one used by R. Williams and M. P. Bryant for culturing bacteria from anaerobic sludge digesters (unpublished data) and is different from 98-5 medium in that glycerol, Trypticase, and hemin are included and mineral solution S2 is substituted for mineral solutions 1 Medium 10 (M10) was developed by Caldwell and Bryant (12) and contains a mixture of volatile fatty acids, hemin, Trypticase, and yeast extract in place of rumen fluid. Supplemented M10 as described by Barnes and Impey (3) contains liver extract (5%, vol/vol) and chicken fecal extract (10%, vol/vol) added to M10. Liver extract (3) was prepared by heating a 13.5% (wt/vol) solution of dehydrated liver (Difco Laboratories) at 50 C for 1 h. The extract was clarified by centrifugation at 10,000 x g, adjusted to neutral pH, sterilized by autoclaving, and stored 15 min) equal quantities (wet weight) of feces and cecal digesta, respectively, and water. The preparations were clarified by centrifugation (10,000 x g, 10 min), neutralized, again autoclaved (15 psi, 15 min), and stored refrigerated. In addition to these culture media, two types of blood agar media were evaluated with respect to recovery of total anaerobes from cecal material: VL blood (oxalated horse blood) agar as described by Barnes and Impey (3) and Schaedler blood (defibrinated rabbit blood) agar of Starr et al. (32). These media were equilibrated and tubed under CO2 gas phase. Blood agar, prepared in this way, supported hemolysis of known hemolytic bacteria and therefore contained intact red blood cells. In the preparation of all roll-tube media, components except Na2CO, buffer, reducing agent (cysteine or cysteine-sulfide), and blood were diluted to volume in round-bottom flasks, equilibrated with CO2 gas by gentle boiling, fitted with rubber stoppers, and sterilized by autoclaving (15 psi, 15 min). After autoclaving, a sterile reducing agent, buffer, or blood was added to media held at 48 C and then dispensed (9-ml amounts) anaerobically into sterile, rubber-stoppered, disposable tubes (18 by 150 mm, Bellco Glass, Inc.). Direct microscope counts, colony counts, and statistical analysis. Direct microscope cell counts were made on cecal samples employing a Petroff-Hauser bacterial counting chamber and a phase-contrast microscope following the method of Meynell and Meynell (25). For microscope counts, cecal sample dilutions of 10-i were made in 0.9% saline containing 10% Formalin. Colony counts in roll tubes were determined after 3, 6, and 14 days of incubation with the aid of a Quebec colony counter. Colony counts from eight roll tubes (prepared from a single dilution) were determined using the colony-counting criteria of Bryant and Robinson (9). Mean counts and standard errors from several cecal samples were obtained, and the data were subjected to an analysis of variance and differences between means evaluated with the 5% least significant difference (31). In some cases, colony counts were converted to percent recovery of the direct microscope counts. Isolation of cecal bacteria and presumptive identification of strains. Fifty isolated colonies were picked from single roll tubes of M98-5 (containing usually 50 to 100 colonies) inoculated with dilutions of cecal contents and incubated for 6 days. About 300 colonies were isolated from single cecal samples of six different birds. Isolates were subcultured onto maintenance slant medium (composition as modified 98-5, Table 1, except glucose, cellobiose, and maltose were at 0.05% wt/vol concentration). Wet mounts of each isolate, prepared from the water of syneresis of slant media (24to 48-h cultures), were observed for morphology, motility, and purity with phase-contrast microscopy. These same cultures were gram stained according to the Kopeloff modification (19). Isolates containing more than one morphological type were separated, and strains were reisolated from roll streaks (19) of M98-5 medium. Pure cultures of strains were grouped and presumptively identified by using media and methods described by Caldwell and Bryant (12). Oxygen relations (facultatively anaerobic or anaerobic) of isolated strains were determined on aerobic plate media. Cultures (by loopful) were streaked onto brain heart infusion-blood agar described by Holdeman and Moore (19), and surface growth was observed at 1, 3, and 6 days of incubation at 37 C. In addition to these preliminary tests, fermentation products elaborated in glucose medium (11) were analyzed for volatile and nonvolatile acids. Selected strains from each of the presumptively identified groups were then processed through carbohydrate fermentation and biochemical tests. Organisms were identified according to classification schemes of Holdeman and Moore (19) and by comparison with other published data on anaerobic bacteria. The basal medium for carbohydrates and substrates fermented in these studies was that of Bryant (11) and contained the following components: 20% (vol/vol) rumen fluid, CRF2; 7.5% (vol/vol) each of mineral solutions 1 and 2; 0.5% (wt/vol) Trypticase; 0.05% (wt/vol) cysteine-hydrochloride; 0.06% (wt/vol) Na2CO; and 0.0001% (wt/vol) resazurin. Most carbohydrates were prepared as 10% wt/vol solutions, filter sterilized, equilibrated with CO2, and added (0.5% final concentration) aseptically to the basal medium. Medium and tests for esculin hydrolysis, gelatin liquefaction, nitrate reduction, and indole production were as described by Bryant and Doetsch (8). All of these media were prepared by the method of Bryant and Burkey (7) and tubed in 4-ml amounts in sterile, rubber-stoppered tubes (13 by 100 mm, Bellco Glass, Inc.) under 10% CO2-90% N. gas mixture. Growth, terminal pH, and biochemical tests were determined on cultures incubated for 7 days at 37 C. The inoculum medium in these studies was the same as M98-5 (Table 1) except glucose, cellobiose, and maltose were at 0.3% (wt/vol), and agar was omitted. All tests were inoculated with 3 drops of a 24-to 48-h inoculum medium culture under 10% CO2-90% N2 gas phase. Analysis of fermentation products. Volatile (acetic, propionic, butyric, and valeric) and nonvolatile (lactic and succinic) acids formed in glucosecontaining media were analyzed by gas chromatographic methods. The gas chromatograph used was a Hewlett-Packard model 5754B equipped with a flame ionization detector. Fermentation media samples (1 ml) were acidified with 0.1 ml of 6 N HCl, and any precipitate formed was removed by centrifugation. Acidified samples were analyzed for volatile acids on a column packed with 20% Carbowax 20 M TPA on Chromosorb W (Varian, 60 to 80 mesh, A/W, DMCS). Nonvolatile acids were methylated in acidified media and analyzed by a modification of the procedure of Hautala and Weaver (18). Formic acid was determined enzymatically by the method of Rabinowitz and Pricer (28). Hydrogen gas produced in gas glucose medium (12) RESULTS AND DISCUSSION Effect of incubation time on colony counts with different roll-tube media. The data in Table 2 show the effect of incubation time on colony counts in 98-5, M98-5, and M10 media. Although the number of colonies continued to increase throughout the 14-day incubation period, the colony counts at 14 days were not significantly higher (P = 0.05) than those at 6 days with M98-5 and M10. In contrast, there was a significant (P = 0.05) increase in colony counts at each incubation time with 98-5. At all intervals of incubation, colony counts were significantly higher in M98-5 than in either 98-5 or M10 (Table 2, last column). Six-day colony counts in 98-5, M98-5, and M10 represented 79%, 92%, and 85%, respectively, of the 14-day counts. When 6-day counts were converted to percent recovery of the direct microscope cell count, 42.8% (98-5), 59.6% (M98-5), and 45.6% (M10) of the total bacterial population in cecal contents could be cultured. Deletion, addition, or substitution of components in M98-5 medium. Omission of rumen fluid from M98-5 reduced colony counts by 50%, whereas deletion of hemin alone made no significant difference in the number of colonies observed (Table 3). Colony counts in M98-5 lacking rumen fluid were similar to those obtained with M98-5 without rumen fluid and hemin. These results indicate that rumen fluid enhances the growth of many cecal bacteria when Trypticase is the only other source of organic growth factors with the exception of the carbohydrate energy sources. From the data, it is not possible to determine whether hemin is a nutritional requirement for cecal anaerobes because rumen fluid would be expected to contain heme (13). Preliminary nutritional studies on representative isolates (48 strains) from each group given in Table 6, however, indicate that few if any of these bacteria require hemin for growth or are stimulated by it. Hemin does augment the growth of most strains of Bacteroides isolated from the bovine rumen (10,13,22), human oral cavity, and intestine (16,24). Colony counts in medium M98-5 were not significantly (P = 0.05) altered when the following single changes were made (data not shown): (i) substitution of minerals S2 with minerals 1 and 2, (ii) deletion of Trypticase or substitution of Trypticase with Casamino Acids (0.2%), (iii) addition of yeast extract (0.05%), and (iv) deletion of glycerol. The reason for M98-5 yielding higher colony counts than 98-5 is not clear, particularly because deletion of Trypticase from M98-5 has no effect on colony counts. Perhaps the added energy source, maltose, or the combined addition of maltose, Trypticase, and glycerol in M98-5 enhances the growth of additional strains of cecal bacteria. Recent unpublished work by E. Barnes 27,1974 on March 18, 2020 by guest http://aem.asm.org/ Downloaded from chicken cecal anaerobes require liver extract to grow in carbohydrate-containing media. In our experiments, addition of liver extract (5 to 20%) to M98-5 (Table 4) did not significantly affect colony counts. To test the possibility that chicken cecal extract might replace rumen fluid for the growth of cecal bacteria, M98-5 (minus rumen fluid) was supplemented with 5 to 30% cecal extract. No increase in colony counts was noted ( Table 4). Also supplementing M98-5 with varying amounts (2.5 to 20%) of cecal extract did not increase counts above the nonsupplemented M98-5 medium. Comparison of various media on percent recovery of anaerobes. In Table 5, the percent recovery of anaerobes in different nonselective media is compared. Although considerable variation (10 to 30%) was observed among cecal samples with all media tested, M98-5 medium gave consistently higher percent recoveries (mean of 60%) than M10, supplemented M10, or blood agar media. Barnes and Impey (3) have found that addition of liver and chicken fecal extracts to M10 is necessary for the isolation of many fastidious anaerobes from the chicken cecum. It may be concluded from our studies that supplementation of M10 with liver extract and/or chicken fecal or cecal extracts yielded 46 to 54% of the direct microscope cell count. These results indicate that such additions only marginally improve the recovery of bacteria b Means not followed by the same letter are significantly different at the 5% level of probability. from that of M10 alone. Moreover, we have consistently observed higher percent recoveries (15 to 25% higher) with M10 or supplemented M10 than those reported by Barnes and coworkers (3,4). With the blood agar roll-tube media tested, 25% of the microflora could be cultured. The high partial pressure exerted by CO2 (in the absence of added Na2CO0), however, altered the pH of VL blood agar to 6.5 and of Schaedler blood agar to 5.9 and, therefore, may have inhibited the growth of some bacteria. In subsequent experiments, six cecal samples were tested on VL blood agar and Schaedler blood agar buffered with Na2CO3 (0.4% vol/vol) to a pH of 6.8 to 6.9. The median percent recovery with VL blood agar was 33% (range 21 to 44%) and with Schaedler blood agar was 44% (range 33 to 52%) of the total microscope cell count. In contrast, the median percent recovery with M98-5 medium in these experiments was 81% (range 72 to 96%). These data indicate that for primary isolation of cecal anaerobes, prereduced blood agar media are inferior to rumen fluid media (M98-5). Also, adequate buffering of blood agar media in roll tubes is important when CO2 is used as a culture gas. In contrast to the culture counts in anaerobic roll tubes, plate counts of total aerobes on Eugonagar or of fungi on Sabouraud agar represented only 2% of the direct microscope counts. Most of the strains isolated on Eugonagar were identified as Escherichia coli and facultatively anaerobic streptococci in further studies. Characterization of cecal strains. From M98-5 medium, 298 strains of bacteria from six cecal samples were isolated. Strains were initially grouped according to a few morphological and physiological features, as well as fermentation products formed from glucose ( Table 6). Identification of selected strains was also based on a comparison of their reactions in various fermentation and biochemical tests to those of known species (Table 7). The predominant microflora is represented by at least 11 groups of bacteria, most of which are gram positive and strictly anaerobic, although two groups of facultatively anaerobic cocci were isolated. Group I was composed of gram-negative cocci or budding coci (1 by 1.5 to 2 Aim), which were clubor dumbbell-shaped cells arranged in pairs and chains. These organisms in a rumen fluid medium (Table 6) were obligately anaerobic and produced small amounts of H2 gas, and formic, acetic, propionic, and butyric acids from glucose. One strain (E10), further analyzed by the VPI Anaerobe Laboratory on peptone-yeast extract basal medium, was aReactions given are those for a majority of the strains within a group. Symbols: oxygen tolerance, A (anaerobic), F (facultative); +, positive reaction; -, negative reaction. Superscripts refer to reactions of a few strains; fermentation products from glucose: f (formic); A, a (acetic); P, p (propionic); b, (butyric); L, 1 (lactic); s (succinic). Upper-case letters refer to acids formed in amounts of 10 ,mol/ml of medium or greater, whereas lower-case letters refer to amounts less than 10 Mmol/ml. Products in parentheses are formed by a few strains. None of the strains tested digested cellulose, and only a few strains in Group VIb were motile. I11a IlIb Ilc IVa IVb IVc V VIa VIb 4 6 5 3 4 23 11 14 9 shown to ferment amygdalin, glucose, maltose, raffinose, and trehalose, to weakly ferment fructose, lactose, and salicin, and to hydrolyze esculin and produce formic and butyric acids from glucose. In preliminary nutritional studies, yeast extract was found to enhance the growth of bacteria in this group. A vitamin mixture containing thiamine-hydrochloride, nicotinamide, riboflavin, pyridoxine-hydrochloride, biotin, folic acid, and DL-thioctic acid could replace yeast extract as a growth stimulant. Foubert and Douglas (15), in a taxonomic analysis of anaerobic micrococci, described a strain (U5) that was isolated from the human uterus. This unnamed strain is similar morphologically and in fermentation characteristics (in peptone-yeast extract basal medium) to our Group I. Strains similar to Group I have also been isolated and described by Gossling (Abstr. Ann. Meet. Amer. Soc. Microbiol., p. 81, 1972) from human feces and by Barnes et al. (5) from chicken cecal contents. Group II was composed of facultatively anaerobic gram-positive cocci (1to 2-um diameter) occurring as singles, pairs, or tetrads. Many strains on initial isolation did not grow on aerobic plates. These strains fermented glucose, fructose, lactose, maltose, and sucrose, reduced nitrate, and formed lactic, acetic, butyric, and formic acids from glucose. They were catalasenegative and therefore differed from catalasepositive species of Micrococcus, Staphylococcus, and Sarcina (1). These organisms do not appear to belong to any previously described genus. Strains in Group III were represented by three different types of gram-positive cocci in chains. Group Ila was comprised of facultatively anaerobic cocci (1 to 1.5 by 1 to 2 ,um) in chains of lancet-shaped cells. None of the strains in Group HIa were hemolytic, but they fermented a wide variety of sugars and may be related to species of Group D streptococci (14). Some strains in this group did not produce an amount of lactic acid characteristic of fecal streptococci and, therefore, could not be species of the genus Streptococcus. Groups II1b and IlIc were similar in morphology and consisted of large gram-positive lancet cocci (1 to 1.5 by 1.5 to 2.5 Mm) arranged in pairs and chains. Similar fermentation products from glucose (acetic and small amounts of propionic and lactic acids) with large amounts of H2 gas were produced by strains of both groups. Group hIb produced H2S and fermented fructose and ribose, whereas Group Ec was weakly fermentative on most sugars. Yeast extract (Groups hIb and mc) and rumen fluid (Group IHb) stimulated the growth of these anaerobic streptococcal types. Groups 11Tb and mc represent species related to Peptostreptococcus Kluyver and van Niel according to emended descriptions of this genus by Rogosa (29). Group HIb may be similar to Hare Group VII isolated from human feces, but an insufficient number of characteristics were given in the publication of Thomas and Hare (33) for a suitable comparison. Strains in Groups hIb and Ec were not related to anaerobic streptococci isolated by Barnes and Impey (3) from poultry ceca or any of the several species of Peptostreptococcus classified by Holdeman and Moore (19) and may constitute new species of Peptostreptococcus. Group 1TIb strains also appear to be closely related (based on similar fermentation properties) to the chain-forming anaerobic streptococci (strain 21-29) isolated by Harrison and Hansen (17) from turkey cecal feces. Group IVa was one of the largest groups of anaerobes isolated and consisted of gram-positive, irregular, pleomorphic rods (1 by 2 to 3 Mlm). Most strains were strict anaerobes, but a few also grew aerobically. These organisms were identified as Propionibacterium acnes according to criteria of Holdeman and Moore (19). A number of variants were observed in this group that could be placed into biotypes A, D, and G as described by Pulverer and Ko (27). Barnes and Impey (4) have also isolated P. acnes from the chicken cecum. The growth of many of our P. acnes strains was enhanced by yeast extract, rumen fluid, and Tween 80. Group IVb consisted of some of the more active strains isolated. These were gram-positive organisms with rounded ends (1 by 2.5 to 4 Mim) occurring as singles, pairs, and long chains. Strains in this group were identified as Eubacterium rectale (19). Group IVb strains were also similar to strain EBG 1/80 isolated by Barnes and Impey (3) from chicken cecal contents. The growth of these strains was stimulated by yeast and cecal extracts and rumen fluid. Group IVc included gram-variable (many stained gram negative in older cultures) fusiform and lancet-shaped cells (1.5 by 2.5 Am) distributed as singles, pairs, and chains. The majority of strains in this group were nonfermentative, but a few hydrolyzed esculin and starch and fermented sucrose. They appear to be related to species of Eubacterium. Group V consisted of large, gram-negative pleomorphic, fusiform-shaped cells (1 to 1.5 by 2.5 to 5 Mm) in pairs and chains. These strains resembled species classified as Bacteroides clostridiiformis by Holdeman and Moore (19). They were characterized by production of lactic and acetic acids from glucose and by having a variable and limited fermentation capacity. Recently, known ATCC strains of B. clostridiiformis have been observed to form spores and are, therefore, species of Clostridium (W. E. C. Moore, personal communication). Spores have not been detected in our cultures of B. clostridiiformis even after prolonged incubation (3 weeks) at least in our rumen fluidglucose liquid medium. Similar strains of B. clostridiiformis were isolated from chicken cecal material by Barnes and Impey (3). Two types of spore-forming rods comprised Group VI. Group VIa contained pleomorphic, nonmotile cells (1 by 2 to 3 Mm) bearing terminal spores. These were similar to known strains of Clostridium ramosum in that glucose, fructose, lactose, maltose, mannose, melibiose, and sucrose were fermented (19). Strains in Group VIb were motile, fusiform rods (1 by 3 to 4 Mm) with subterminal spores and were species of Clostridium that could not be identified. Distribution of bacteria in the cecum. Results of this work indicate that 90% of all strains isolated from cecal material of six animals (5 weeks old) consist of at least 11 groups and subgroups of facultative and anaerobic bacteria (Tables 6 and 7). Approximately 10% of the strains were made up of unknown species (miscellaneous rods) that could not be identified. The predominant groups that were consistently isolated from high (10-9) dilutions of samples were distributed as follows: 7% as gram-negative budding cocci (Group I, unknown species); 12% as gram-positive facultative cocci (Group II, unknown species); 14% as streptococci (Groups IIIa, b, c; Streptococcus and Peptostreptococcus); 32% as gram-positive rods (Groups IVa, b, c; P. acnes, E. rectale and Eubacterium sp.); 14% as gram-negative rods VOL. 27,1974 68a005 on March 18, 2020 by guest http://aem.asm.org/ Downloaded from (Group V, B. clostridiiformis); and 10% as spore-forming rods (Group VIa, b; C. ramosum and Clostridium sp.). Barnes and Impey (3), on the other hand, found that the anaerobic cecal microflora of the 5-week-old chicken was composed of 40% grampositive nonspore-forming rods and bifidobacteria, 40% gram-negative rods (Bacteroidaceae), and 15% strains of peptostreptococci and curved rods. Not only are there these differences in the distribution of bacterial types in our study and that of Barnes and Impey (3), but the latter investigators also isolated gram-negative rods such as B. fragilis and B. hypermegas. Moreover, bifidobacteria and lactobacilli, cultured from chicken intestinal contents by some investigators (26,30,34), were not recovered in any sample analyzed in this study, at least in high dilutions (10-9) of cecal material. We isolated similar strains of budding cocci, P. acnes and B. clostridiiformis, as did Barnes and co-workers (4,5). It may be concluded, therefore, that the relative proportion of the various bacterial groups within the intestinal microbial population of chickens are somewhat variable owing to such differences as breed, diet, growth conditions, and geographic location of animals. The relative proportion of bacterial groups would also be affected by the isolation methods used by different workers.

v3-fos

2020-12-10T09:04:22.874Z

1974-07-01T00:00:00.000Z

237234001

s2

Fate of Ochratoxin A and Citrinin During Malting and Brewing Experiments The fate of ochratoxin A and citrinin during malting and brewing processes was studied by the use of naturally contaminated lots of barley, as well as by the addition of crystalline toxins to the mash. Complete degradation was observed for ochratoxin A from moderately contaminated barley lots and for citrinin added to mash. The use of highly contaminated barley resulted in transmission of ochratoxin A into the beer, but only 2 to 7% of the initial content was detected, corresponding to levels of 6 to 20 μg of ochratoxin A per liter of beer. Barley lots with this high ochratoxin contamination (1,000 to 5,000 μg/kg) will be easily detected and, therefore, because of pronounced deterioration, should be rejected during inspection upon admittance to the breweries. Ochratoxin A and citrinin are mycotoxins with nephrotoxic properties, and they have been found as causal determinants in a naturally occurring kidney disease, porcine nephropathy (6). Kidney lesions have been induced experimentally in many animal species, and it is reasonable to expect the toxins to act on human kidneys as well. Ochratoxin A as a natural contaminant was first reported from the U.S.A. (12), where a sample of maize was found to be contaminated with approximately 150 Mg of ochratoxin A per kg. In inspection of maize from different regions of the U.S.A., ochratoxin A has repeatedly been found at levels of from 83 to 166 Mg/kg (13,14). This mycotoxin has also been detected in American barley samples (S. Nesheim, Annu. Meet. Ass. Offic. Anal. Chem., 85th, Washington, D.C., Abstr., 1971) at levels of 12 to 38 Mg/kg. Ochratoxin A was found in concentrations of 30 to 27,000 Mg/kg in 18 out of 29 samples of Canadian grain stored under damp conditions (11). The cereal samples consisted mainly of wheat but also included oats, barley, and rye. Contamination of Canadian wheat with ochratoxin A at levels of 20 to 100 Mg/kg was previously reported (10). Thirteen of the above mentioned samples of heating grain were simultaneously contaminated with citrinin at levels of 70 to 80,000 Mg/kg. The citrinin-contaminated samples included wheat, oats, barley, and rye. In Denmark, ochratoxin A-contaminated samples of barley and oats have frequently been observed at levels of 28 to 27,500 Mg/kg (6). Some of these samples were simultaneously contaminated with citrinin (160 to 2,000 Mg/kg). In Sweden, barley and oats have been found to be contaminated with ochratoxin A at levels of 16 to 410 Mg/kg (5). Most of the cereal samples reported here have been collected from lots which were intended to be used as animal feed. The breweries perform careful control of barley and other cereals before acceptance for malting and brewing purposes, because moldy cereals will spoil the flavor and other qualities of the beer, e.g., by causing gushing (3). In spite of the control, contaminated lots of cereals may pass, because organoleptical changes are minor and germination tests and microbiological control cannot prevent the passing of low-contaminated lots, which can only be detected by chemical analysis for mycotoxins. The fate of ochratoxin A and citrinin during the malting and brewing processes is therefore of considerable interest from a food hygiene and food safety point of view. (Some of the results presented here were included in a report at the 14th Congress of the European Brewery Convention, Salzburg, May 1973.) MATERIALS AND METHODS Barley. Lots naturally contaminated with ochratoxin A and/or citrinin were collected from farms, where the lots had caused kidney disease in pigs (6). Malting barley, of normal high quality, was obtained from the Carlsberg Breweries and used as uncontaminated barley (control). Malting and brewing. (i) Micromalting. Portions (70 g) of barley were steeped in 250-ml plastic beakers with 12 holes (2-mm diameter) in the bottom. Alternating periods of 8 h with water and 16 h without water were applied at 12 C. Steeping was finished when a moisture content of 43 to 44% was obtained, and germination was carried out in the same beakers at 12 C. Once a day the barley was taken out for inspection, and the kernels were loosened from each other. Total steeping and germination time was 9 days. The samples were kilned in nylon bags for 8 h at 45 C and 16 h at 75 C. (ii) Experimental brewing. Ten kilograms of malt was sprayed with 400 g of water and ground 10 min later. The mashing was an infusion system starting with 35 liters of water at 39 C. After 40 min, the temperature was raised to 51 C for 60 min, and 15 g of lactic acid was added. Eight minutes later, the mash was heated 1 C per min to 65 C, and after 40 min of saccharification the temperature was again raised in 1-C increments per min to 75 C and then held for 30 min. The mash was transferred to the lauter tun for filtration and sparging with 44 liters of water. The wort was boiled with hops for 90 min, cooled, and aerated. The wort was distributed into two vessels, each containing 25 liters of wort, and was fermented with 90 g of centrifuged yeast in each vessel at 10 C for about 7 days. After racking, the beer was stored at 5 C for 1 week and at 0 C for 4 weeks, after which it was filtered, carbonated, bottled, and pasteurized. (iii) Experimental brewing with barley and enzymes. Five kilograms of barley was steeped for 30 min at 50 C. The steep water was used later for mashing. After air-drying, the barley was ground and mashed in 20 liters of water at 50 C with addition of 5 g of bacterial amylase (BAN). The temperature was raised to 80 C in 60 min, and, after 30 min at this temperature, the barley-mash was pumped to the malt-mash prepared from 5 kg of ground malt and 17 liters of water at 20 C. To the mixed mash at 50 C was added 5 g of BAN and 5 g of bacterial proteinase (BPN). BAN (EC 3.2.1.1) and BPN (EC 3.4.4.16) were both obtained from Bacillus subtilis. The enzyme preparations were obtained from Novo, Copenhagen. After 30 min at 50 C, the temperature was raised in 1-C increments per min to 63 C, which was held for 45 min. Mashing was finished at 75 C, and the brewing was continued as described above. Germination of the barley was determined (2), and the following analyses were carried out on the wort. Extract concentration (% Plato) (1), attenuation (degree of fermentation) (1), nitrogen (7), viscosity by use of Hoppler viscosimeter, and color were measured in a spectrophotometer at 465 nm in a 1-cm cell. Mycotoxin analysis. (i) Solid samples. Samples (50 g) of solid material (barley, malt, spent grains) were ground and water was added and, after acidification, they were extracted with chloroform. Quantitation of citrinin (4) and ochratoxin (8) was carried out by the use of densitometer techniques. (ii) Liquid samples. Samples (350 ml) of liquid material (wort, beer) were acidified and extracted with chloroform. Quantitation was made as above. During the investigations, the following experiments were carried out: RESULTS Ochratoxin A. Moderately contaminated barley samples were used during experiment I, and, although the germination percentage of lot MT 480 was a little below the normal specification, a reasonable malting experiment could be performed. The results of ordinary malt analyses were normal, although there was a moldy smell during the malting, and no ochratoxin was detected in the malt (Table 1). Two heavily contaminated lots of barley were used for experiment II (experimental brewing). The lots had a moldy smell, very pronounced in MT 100, and because of a very low germination percentage, malting was out of the question, and brewing could be carried out only with addition of bacterial enzymes. As indicated in Table 2, there was a strong degradation of ochratoxin A during the brewing process, with the final beer containing 2 to 7% of the initial amount of ochratoxin A, resulting in a concentration of 11 to 20 gg/liter. Spent grains contained a proportional higher level, with a concentration of 80 to 210 ug/kg. The normal wort analyses (Table 3) showed an increase of soluble nitrogen and more pronounced color. The smell of the wort and the flavor of the beer was abnormal, especially in brew no. 1321. To show whether barley-malt enzymes had a different effect on the degradation than did bacterial enzymes, experiment III was conducted, with the addition to normal malt of crystalline ochratoxin A. A similar degradation, as in the previous experiment, was observed ( Table 2) with 4% of the initial amount found in the beer, equal to 6 gg/liter. On the thin-layer chromatography plates, fluorescent spots different from ochratoxin A and ochratoxin a were present in the wort but absent in the final beer. Citrinin. Malting and brewing experiments were intended to be done with the use of a naturally contaminated lot of barley. However, the germination percentage was zero, and no malting was possible. Instead, crystalline citrinin (10 mg) was added to normal malt (experiment IV), and no citrinin could be detected in the wort and spent grains. DISCUSSION The malting process completely degrades ochratoxin A present in moderately contami-nated barley lots. When heavily contaminated lots are used for mashing, a pronounced reduction of ochratoxin A takes place during the process, indicated by the presence in the wort of only 11 to 19% of the initial toxin content in the barley. The subsequent fermentation process degrades ochratoxin further, so that only 2 to 7% of the initial content is present in the beer, corresponding to 6 to 20 ug/liter. However, the barley used to produce this level in the beer was so heavily contaminated and deteriorated that similar lots would not be used by breweries. Citrinin degraded at an even faster rate than ochratoxin during the mashing process and was not present in detectable amounts in the wort. Only the use of highly contaminated lots of barley will result in production of ochratoxincontaminated beer. As these lots will be rejected during inspection upon admittance to the breweries, the problem of transmission of these mycotoxins from cereals to the beer seems very unlikely. The limitation of the present investigations is the use of TLC techniques only for toxic metabolite detection; no biological test systems for detection of possible toxic, nonfluorescent break-down products of ochratoxin A and citrinin were employed. LITERATURE

v3-fos

2018-04-03T03:47:12.648Z

2009-02-12T00:00:00.000Z

5325991

s2

Sources of Resistance to Powdery Mildew in Barley Landraces from Turkey : Powdery mildew on barley, caused by the pathogen Blumeria graminis f. sp. hordei, occurs worldwide and can result in severe yield loss. Germplasm of barley, including landraces, commercial cultivars, wild relatives and breeding lines are stored in more than 200 institutions. There is a need for characterization of this germplasm in terms of resistance to biotic and abiotic stresses. This is necessary in order to use specific accessions in breeding programs. In the present study, 129 barley landraces originated from Turkey and provided by the ICARDA genebank were tested for resistance to powdery mildew. Seedling resistance tests after inoculation with 19 differentiated isolates of B. graminis f. sp. hordei were used to postulate the presence of resistance genes. From the 129 landraces studied, plants of 19 (14.7%) of them showed resistance to infection with powdery mildew. Based on preliminary tests from these 19 landraces, 25 resistant single plant lines were selected for testing with differential powdery mildew isolates. Seven lines were resistant to all 19 isolates used. However, only one line (5583-1-4) showed resistance scores of zero against all isolates used. It is likely that this line possesses unknown, but highly effective genes for resistance. In five resistant lines it was not possible to postulate the presence of specific resistance genes. In 19 lines the presence of the genes Mlp , Mlk , Mlh , Mlg , Ml(CP) , Mlat , Mla3 , Mla6 , Mla7 and Mla22 were postulated. These new sources of highly effective powdery mildew resistance in barley landraces from Turkey could be successfully used in breeding programs. Introduction Barley (Hordeum vulgare L.) is an economically important cereal crop which is known to be drought, cold, and salt tolerant and well-adapted to low-input environmental conditions [1,2]. It is cultivated at high altitudes and commonly under rain-fed conditions. It is often grown in marginal agricultural areas with low annual precipitation, often less than 220 mm [3]. Barley ranks as the fourth crop in the world, after wheat, maize and rice, in terms of the area of cultivation. Almost half of the world's barley area is in Europe, where barley is second crop after wheat in cultivated area [4]. Germplasm of barley, including landraces, commercial cultivars, wild relatives and breeding lines is very diverse and is stored in more than 200 institutions [5,6]. Barley landraces are an important source of genetic variation and resistance to biotic stresses including powdery mildew [7][8][9]. Turkey is characterized by the presence of diverse agroecological zones and a long history of agriculture. It is known to be a rich source of barley landraces. They are still planted in this country, and they are characterized by a high level of resistance to biotic and abiotic stresses. There is a need for characterization of this germplasm in terms of resistance to biotic and abiotic stresses. This knowledge is necessary in order to use specific accessions in breeding programs [10][11][12]. Effective controlling of barley powdery mildew is possible by growing genetically resistant barley cultivars. This method of crop protection is relatively inexpensive and it is environmentally friendly. These cultivars started being used from the beginning of the application of modern, intensive methods in barley production because these production methods created favorable conditions for development of this disease [25][26][27][28][29][30]. Currently, powdery mildew of barley is one of the most common and most widespread disease of barley in Europe and another barley regions of the world, causing significant yield losses [20,21,31]. When a cultivar containing one dominant resistance gene is grown on a large acreage, new virulent B. graminis races may occur within 4-5 years. Exceptions are recessive genes for resistance such as mlf and mlo. However, many factors, e.g., temperature, water stress or light intensity, may affect the use of these genes in breeding programmes [20,22,28]. At least 38 different genes/alleles have been used in varieties grown in Europe [32]. Nevertheless, barley breeders, geneticists and plant pathologists are constantly looking for new, efficient sources of powdery mildew resistance, in order to combine them with those already used in modern cultivars, and to increase their resistance durability [31,33,34]. Most of the original sources of powdery mildew resistance genes came from domesticated cultivars in Europe [25,26,35]. These sources of resistance were easy to be used in breeding but the number of resistance genes was limited. Breeders and geneticists have been looking for new sources of resistance in non-European germplasm. Most of these studies were conducted using collections of landraces and differential sets of powdery mildew isolates [36,37]. Previous studies showed that barley landraces from Turkey are rich sources of genetic diversity for plant breeding, including resistance to pathogens [10][11][12]38,39]. Pathogen Nineteen differential Bgh (B. graminis f. sp. hordei Em Marschal) isolates with virulence genes corresponding to known resistance genes were used ( [42], provided by Dr. L. Munk (Royal Agricultural and Veterinary University, Copenhagen, Denmark) and on 8 additional cultivars. Landraces and Single Plant-Lines Resistance Tests First, samples of 30 plants from each of the landraces were tested with the Bgh 33 isolate (the most avirulent one) under controlled chamber conditions with a 16/8 h day/night photoperiod and a 22/16 • C temperature regime. Seedlings with a fully expanded first leaf were inoculated with Bgh isolate by shaking conidia from the susceptible cv. Manchuria. After 8-10 days, infection types were scored. Plants with disease scores of 0 to 1 were classified as highly resistant (R), plants that scored 2 as a moderately resistant (M) and rating of 3 or 4 as susceptible and very susceptible (S). Plants with the score 0(4) possess a resistance gene in locus Mlo. The cultivar Manchurian CI 3230 was used as a susceptible control. Based on the results of this preliminary experiment, 25 resistant single plant lines from 19 landraces were selected. A highly resistant reaction type was observed on 13 lines, and a moderately resistant reaction type was observed on 11 lines. In 5 landraces, segregation of RT was observed (Table 3). Next, they were grown in greenhouse conditions to obtain seeds for future evaluations using a set of 19 Bgh differential isolates. Postulation of resistant genes in tested lines was based on a comparison of reaction spectra observed on tested plants and the barley differential set infected with differential Bgh isolates (Table 1). This was performed on the basis of the gene-for-gene hypothesis [44]. Table 2. B. graminis f. sp. hordei isolates used for artificial inoculation and their virulence spectra against resistance genes on differential set of Pallas near-isogenic lines and 8 cultivars. No. Results Plants of 19 (14.7%) of the tested landraces were resistant to infection with Bgh33 isolate in preliminary testing. In five landraces, segregation of RT was observed. Based on preliminary tests from these 19 landraces, 25 resistant, single plant lines were selected for testing with differential isolates. From these lines, seven were resistant to all 19 isolates used. However, only one line (5583-1-4) showed resistance scores of zero against all isolates used (Table 3). In five lines it was not possible to postulate the presence of specific resistance genes. In 19 lines, the presence of the genes Mlp, Mlk, Mlh, Mlg, Ml(CP), Mlat, Mla3, Mla6, Mla7, Mla22 and unknown genes (genes not present in differential set) were postulated. Discussion Barley landraces from Turkey are a rich source of genetic diversity for plant breeding, including resistance to powdery mildew [10][11][12]38,39]. This was confirmed in the presented study. Single plant lines selected from 19 (14.7%) of the tested landraces were resistant to infection with powdery mildew. Resistant lines selected from landraces are a very valuable material for resistance breeding. This kind of germplasm is the simplest source of resistance to use directly in breeding programs. Because of their adaptability to a wide range of conditions, barley landraces are recognized as an important genetic resource for tolerance and resistance to biotic and abiotic stresses. They carry unique traits and are considered a rich resource for resistance breeding and for the expansion of the gene pool [45,46]. Turkey is a rich source of barley genetic diversity because of its geographic location. The south-eastern region of Turkey is at the top of the Fertile Crescent of the Near East, within the centre of origin of cultivated barley [39]. Barley is one of the oldest cultivated plants grown in Anatolia, and it is the second most important cereal crop following wheat. In addition, in Turkey, the ancestor of cultivated barley, Hordeum spontaneum C. Koch, grows naturally, and powdery mildew epidemics occur in the western and southern parts of the country [12]. All these factors lead to conclusion that coevolution of barley powdery mildew was occurring in Turkey for very long time, and that barley landraces from Turkey may be a rich source of resistance to powdery mildew. This was confirmed in the presented study, in which many resistance genes were identified in lines selected from Turkish landraces. The genetic diversity of barley landraces offers many traits for barley breeding, especially concerning resistance to biotic and abiotic stresses [3,33,[47][48][49][50]. The genetic heterogeneity within the barley landraces is due to a low level of outcrossing occurring in barley, and farmers' management of seed [40,[51][52][53]. This genetic heterogeneity was also observed in the presented study, in which five landraces showed segregation of RT. Seedling resistance tests were used in order to describe infection types expressed by barley lines after inoculation with differentiated isolates of B. graminis f. sp. hordei. This kind of testing is sufficient for disease-resistance screening. It is used commonly in breeding programmes to postulate the presence of specific resistance genes in modern cultivars and to screen germplasm for new sources of effective resistance [36,40,75,76]. However, these kinds of tests are not very useful for identifying and describing partial resistance. For a description of partial resistance there is a need to conduct measurements of resistance characteristics, in addition to the infection type. Furthermore, partial resistance is generally better expressed at the adult plant stage [26,34,77]. Newly identified sources of powdery mildew resistance in 25 single plant lines (originated from 19 landraces) are valuable for barley breeding for resistance. In five lines it was not possible to postulate the presence of specific resistance genes. In 19 lines the presence of the genes Mlp, Mlk, Mlh, Mlg, Ml(CP), Mlat, Mla3, Mla6, Mla7 and Mla22 and unknown genes (not present in differential set) were postulated. Interestingly for barley resistance breeding, seven lines selected from four landraces were resistant to all 19 isolates used in this study. However, the most interesting point from a breeders' point of view was line 5583-1-4, which showed resistance scores of zero for all isolates used. Most probably this line possesses unknown, yet very effective genes for resistance. Future work will include the genetic study of resistance identified in seven single-plant lines by conducting appropriate crosses and the use of molecular markers [58,59,78,79]. Authors intend to introduce these alleles into elite cultivars of barley to create initial materials for European breeding programmes. This is a necessary step between barley genebank collections and the practical use of barley genetic resources in breeding programmes. The new sources of highly effective powdery mildew resistance described in this study could be successfully used in barley breeding programs.

v3-fos

2018-04-03T03:26:13.772Z

1974-06-01T00:00:00.000Z

5808126

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Ultrastructure of a Thermotolerant Basidiomycete Possibly Suitable for Production of Food Protein The imperfect cellulolytic fungus Sporotrichum pulverulentum, which is commonly found growing in wood-chip piles, was grown in submerged culture on wheat shorts and other cereal flours. These substrates were broken down in 1 to 4 days at 30 to 40 C, and the mycelial mass was easily harvested by filtration. Scanning electron micrographs of hyphae in mycelial pellets are presented, and thin sections of conidia and hyphae were studied in a transmission electron microscope. Dolipores in septa of hyphae were observed, and cell walls are shown to be lamellar, which is characteristic of the Basidiomycetes. Actively growing hyphae are full of cytoplasm with numerous mitochondria, whereas old mycelial pellets contain highly vacuolated and almost empty cells. Large quantities of fruit bodies (sporophores) of many kinds of Basidiomycetes have long been eaten because they are nutritious and good tasting. Some higher fungi can be grown in submerged culture, but most of them grow slowly in conventional fermentors and only a few have been used for industrial production of food products (12). Other filamentous fungi, classified either as Ascomycetes or Fungi Imperfecti, are presently being investigated as unconventional sources of protein (13), and commercial production of "mycoprotein" by cultivation of a Fusarium sp. has recently been started (9). Before large-scale production of fungal biomass for human consumption is considered, it is necessary to prove that the product is nontoxic. Information on the systematic position of the organism studied is then useful. It is also important to determine how culture conditions influence the chemical composition and morphology of the organism. The protein content may vary considerably depending on culture conditions, but the nucleic acid content of fungi is usually lower than that of many bacteria, yeasts, and algae (13). Single-cell organisms may be easier to grow in submerged culture than fungi, which have a filamentous or pellet form of growth, but the latter can often be harvested by simple filtration. We have for some years studied the microbial degradation of cellulose and related polysaccha-rides. As part of this project, we have investigated the possibility of growing cellulolytic microorganisms on flours and meals of various cereals and other seeds. One such material is wheat bran, which contains 15 to 17% protein and about 70% carbohydrates, about 80% of which is cellulose and hemicellulose. Very large quantities of bran and other milling fractions containing bran are at present either used only as low-quality animal feed or wasted. The same applies to such products as rice bran, and it would be important if they could be converted to products of higher nutritional value. We have found that a thermotolerant, cellulolytic fungus can grow on various cereal flours, and preliminary experiments indicate that the mycelium has a high nutritional value. The organism we used is a wood-destroying, imperfect fungus that was isolated from a pile of wood chips by Nilsson (6). He found that it caused typical white-rot decay, and the organism was first classified as a Chrysosporium sp., but it has now been found (J. A. Stalpers, personal communication) to be identical with a fungus described as Sporotrichum pulverulentum by Novobranova (7). Its cellulolytic enzymes have been studied by Eriksson and Rzedowski (3), who purified several exo-and endoglucanases and other carbohydrases from cell-free fluids of submerged cultures of the fungus. This report describes some features of the ultrastructure of S. pulverulentum and effects of cultivation time on its cytoplasm. Studies on the effect of culture conditions on its growth morphology and chemical composition and the results of pilot-scale cultivation experiments are presented elsewhere. MATERIAL AND METHODS Fungal strain. The strain of S. pulverulentum that we studied was isolated in 1964 by Thomas Nilsson, College of Forestry, Stockholm, where it is maintained in the culture collection as strain P 127-1. It has also been deposited at Centralbureau voor Schimmelcultures, Baarn, The Netherlands, where it has the number CBS 671.71. Cultivation methods. Stock cultures were maintained on malt extract agar (Difco), on which large numbers of white conidia are formed. Scanning electron microscopy. Freeze-dried cell material was coated with evaporated gold before examination in a JSM-U3 scanning electron microscope operating at 25 kV. Transmission electron microscopy. Mycelial pellets from submerged cultures and pieces of agar containing hyphae were prefixed for 24 h in 2.5% glutaraldehyde buffered with Veronal-acetate buffer, pH 7. The material was further fixed in 2% potassium permanganate in water (4). After dehydration in a graded ethanol series, the material was impregnated with the plastic resin Epon 812, and this was polymerized at 60 C. Thin sections were cut with a diamond knife on an LKB ultramicrotome, and sections were post-stained with 2% aqueous uranyl acetate for 20 min at 60 C and with 6% aqueous lead citrate for 2 min at room temperature. Specimens were examined in a JEOL 100 B electron microscope operating with a double condensor at 60 kV. RESULTS Growth in liquid cultures. Conidia from agar cultures were used to inoculate Fernbach flasks containing a shallow layer of mineral salts medium with 1 or 2% wheat shorts. The cultures were incubated on a rotary shaker (150 rpm) at 30, 35, or 40 C. The spores germinated and formed small hyphal aggregates after about a day. In this medium and under these culture conditions, the small aggregates grew out into spherical pellets which increased in size during the following day with a concomitant decrease in pH to between 4 and 5. The pellets could easily be filtered off on a 30-mesh sieve giving an almost clear, slightly yellow filtrate which had a pleasant smell reminiscent of apples. The maximal growth temperature was between 40 and 45 C, but the fungus cannot be called thermophilic because it also grew below 25 C. Other milling fractions of wheat could be converted to fungal biomass at rates which depended on the fineness of the flour. It took 4 to 5 days at 35 C to degrade coarse wheat bran, whereas fine flours of rye and barley were degraded in a couple of days. Defatted rice bran, which consists of very fine particles, supported rapid growth and was completely degraded in about 2 days. Figure 1 illustrates a sample of freeze-dried mycelial pellets obtained on wheat shorts. Some small bran fragments were undegraded at the time of harvesting, but these could be washed away through a 30-mesh sieve. Growth on whole wheat flour gave smaller (1 to 2 mm), irregular hyphal aggregates, whereas very smooth, 4to 5-mm pellets were obtained on wheat bran. Electron microscopy. Figure 2 is a scanning electron micrograph of the interior of a mycelial pellet harvested from a 3-day-old culture on wheat bran. The mycelium is highly branched, and it can be seen at higher magnification ( Fig. 3) that many of the hyphae have collapsed to flat, fiber-like structures during the freeze-drying. Few conidia are formed in liquid cultures unless these are incubated for a long time or left standing at room temperature for some days. Figure 4 is a thin section of a young conidium attached to a hypha by means of a stalk cell which contains a supporting structure near the conidium. A cap-like structure can also be seen on top of the conidium, and its cell wall is still rather thin. When observed in the light microscope, most conidia are spherical, but some of them are dumbbell shaped (Fig. 5). This mature conidium has a thick, multilayered wall measuring almost 1 Mm, and it contains several nuclei and numerous mitochondria. Hyphae have thinner walls than the conidia, and old cells are often highly vacuolated. Figure 6 shows a thin section of a young hypha grown on malt agar. There is a distinct dolipore between the two cells and a peculiar membranous structure near the septum. The insert in Fig. 6 shows that the hyphal wall has a distinct lamellar structure, and pictures of old hyphae showed that these lamellae occasionally become partly separated from each other. Samples of small mycelial aggregates from 1 to 2 days old submerged on wheat shorts cultures contained a large proportion of hyphae which had an appearance similar to that of the hypha shown in Fig. 6, but very few of the septa had typical dolipores. Cultures which had been grown for more than 3 days on wheat bran contained 4-to 5-mm pellets in which the cells were more or less autolyzed. Figure 7 shows a thin section of some hyphae in a pellet grown on wheat bran for 5 days. DISCUSSION The increasing global demand for high-quality proteins for human food and animal feed has stimulated much work on large-scale production of microbial cells. Single-cell organisms are often easier to grow in submerged culture than filamentous fungi, but they are more difficult to harvest, and many of them contain so much nucleic acid that they cannot be used for human consumption. Many higher fungi are already accepted as a source of food in many parts of the world, but most of them grow slowly at rather low temperatures. Worgan (13) has recently discussed the relative merits of different substrates and general problems in the production of microbial protein. Cellulolytic fungi have earlier been tried for the conversion of paper and various agricultural wastes to animal feed (1,8,10), but many cellulolytic organisms grow very slowly or not at all on lignified plant material. Expensive pretreatments are therefore necessary in order to make such raw materials degradable. Some of the fungi tested, such as Trichoderma viride, Aspergillus fumigatus, and various Fusarium sp., may also produce mycotoxins under certain conditions. The fungus that we studied has some properties which suggest that it may become an interesting new source of protein. It grows rapidly at relatively high temperatures and can, degrade a variety of cheap carbohydrates such as cellulose, hemicellulose, and starch, and it can even grow on lignified tissues. Its systematic position remains somewhat uncertain as long as it must be classified among the Fungi Imperfecti, but our electron micrographs show that it must be an imperfect conidial stage of a basidiomycete. The presence of the characteristic dolipores in the septa of hyphal cells is thus a characteristic feature of Basidiomycetes (2). The lamellar structure of the hyphal walls is also considered to be typical of this group of fungi (5). Furthermore, only Basidiomycetes are known to cause white-rot attack on wood. It is possible that S. pulverulentum is closely related to the higher fungi of the family Polyporaceae, of which many are eaten. We have as yet only carried out preliminary feeding experiments with mycelium grown on wheat shorts, but no harmful effects were noticed when the fungus was given as sole source of protein to young rats, and their rate of growth was normal for over 3 weeks. More extensive feeding tests must, of course, be carried out to prove that the fungus is wholesome. Brans and other milling fractions of cereals contain cellulose, hemicellulose, and starch in varying proportions, and they are interesting raw materials for mycoprotein production. They are available in very large quantities and can easily be stored. Several technical problems must be solved before a fungus can be grown on a large scale on such partly insoluble substrates. Many fungi grow either in filamentous or pellet form, depending on the nature of the inoculum, composition of the medium, or physical conditions (11). It is important to be able to control these factors so that a maximal protein content is obtained in the mycelium. Our electron microscope studies show that large pellets of S. pulverulentum contain a high proportion of partly autolyzed cells, and it has been found that the protein content of these cells is only about 20%. It is therefore obviously desirable to grow the organism under conditions which favor the formation of small hyphal aggregates.

v3-fos

2020-12-10T09:04:22.836Z

1974-09-01T00:00:00.000Z

237231756

s2

Effect of Dissolved Oxygen, Temperature, Initial Cell Count, and Sugar Concentration on the Viability of Saccharomyces cerevisiae in Rapid Fermentations By using 7 × 108 cells of Saccharomyces cerevisiae per ml with which 25° Brix honey solutions were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C, i.e., rapid fermentation, the death rate was found to be high, with only 2.1% of the yeast cells surviving at the end of 3 h under anaerobic conditions. As the dissolved oxygen in the medium was increased from 0 to 13 to 20 to 100% in rapid fermentations at 30 C, there was a progressive increase in the percentage of cells surviving. The ethanol production rate and total were not seriously affected by a dissolved oxygen concentration of 13%, but fermentation was retarded by 20% dissolved oxygen and still further decreased as the dissolved oxygen content reached 100%. When the fermentation temperature was decreased to 15 C (at 13% dissolved oxygen), the rate of fermentation decreased, and the fermentation time to 9.5% ethanol (wt/vol) increased to 6 h. It was found that the higher the temperature between 15 and 30 C, the greater the rate of death as initial cell counts were increased from 1.1 × 107 to 7.8 × 108 cells per ml. At the lowest level of inoculum, 1.1 × 107 cells per ml, there was actual multiplication, even at 30 C; however, the fermentation was no longer rapid. The addition of 15% sugar, initially followed after an hour by the remaining 10%, or addition of the sugar in increments of 2.5 or 5% yielded a better survival rate of yeast cells than when the fermentation was initiated with 25% sugar. h or less. For example, Steinkraus (11) reported that at a temperature of 25 C, honey solution or grape juice (250 Brix sugar concentration) was fermented to 9.5% (wt/vol) ethanol within a 4-h period. The high rate of ethanol production was achieved primarily by using a yeast concentration of 3 x 109 cells per ml. A fermentation time of 6 h or less makes continuous fermentation feasible. However, a problem that severely limits the possibility of applying rapid fermentation to continuous fermentation has been the high death rate of yeast cells. For continuous fermentation, it is essential that yeast viability (as a percentage of the initial yeast charge) remains at 100% in rapid fermentation, and it is desirable that some low level multiplication occurs continuously to replace dead cells and to replace those lost during centrifuging of the product and during the recycling of cells to the fermentor. Although there have been numerous studies on the effect of temperature on growth and death of cells, some having dealt with the effect of temperature on viability of yeast suspensions (14,15), specific information regarding the effect of temperature on viability during rapid alcoholic fermentation appears to be lacking. At incubation temperatures of 32 C and higher, Saccharomyces cerevisiae, Steinberg strain 618, failed to achieve as high alcohol concentrations in a 250 Brix clover honey medium as it did at lower fermentation temperatures ' (11). The role of oxygen in alcoholic fermentations has been reviewed (7). Experience has demonstrated that air or oxygen is essential for good brewery fermentation. Oxygen acts primarily as a terminal acceptor of electrons from the respiratory chain. It also acts as a yeast growth factor. Oxygen appears to be involved in the synthesis of oleic acid and ergosterol, which stimulate yeast growth under anaerobic conditions (1). In brewery fermentation, 20% oxygen saturation of the wort prior to pitching resulted in the maximal production of yeast cells (7). Above 383 that level, increases of cell mass were small, and below that level production of yeast cells was directly related to oxygen supplied. It may be that the yeast cells have the ability to retain the effect of oxygen within the cell and use it anaerobically later (2). The amount of ethanol produced during fermentation is independent of the amount of oxygen supplied prior to pitching. A high degree of aeration results in an increase of biomass, i.e., multiplication and less ethanol production (8). Brettanomyces claussenii showed a higher rate of ethanol production in air than in nitrogen (M. T. J. Custers, Ph.D. thesis, Delft Univ. of Technology, Delft, The Netherlands, 1940). The term ''negative Pasteur effect" was introduced for the phenomenon to emphasize the striking contrast to the well-known Pasteur effect. A similar phenomenon was reported in intact cells of S. cerevisiae (16). Generally, when yeast multiplication is desired, as with bakers' yeast, aeration is used and sugar is added in small increments. This eliminates repression of cell growth by high sugar concentration and insures oxygen availability to the cell. Yeast in a glucose medium consumed oxygen at a very slow rate until glucose was nearly all used (13). Thus, growth in the presence of glucose took place as though the yeast was under anaerobic conditions. Bakers' yeast ferments more than 80% of the sugar to ethanol in spite of vigorous aeration (5). Thus, the classical Pasteur effect does not appear to operate under conditions of yeast growth. Slonimski (9) called this the counter-Pasteur effect. It describes the relationship between the rate of synthesis of respirating enzymes and the rate of fermentation. Yeast growing aerobically in glucose medium does not rapidly synthesize its respiratory enzymes (3). Most of the energy for growth comes from glycolysis. The counter-Pasteur effect is one aspect of what used to be called the glucose effect and is now described as catabolite repression (6). This is the inhibitory effect of glucose on the synthesis of enzymes, which do not immediately contribute to the growth of the cell. At glucose concentrations above 3%, cellular mechanisms for the formation of nucleic acids and proteins are saturated. Consequently, the cell becomes rich in adenosine 5'-triphosphate and intermediates such as pyruvate. The cells, therefore, have no need to tap any extra energy from the citric acid cycle, and to prevent unnecessary tapping of energy the biosynthesis of respiratory enzymes is repressed. The level of aeration supplied to the yeast before or during fermentation is related to the viability of the yeast. There appears to be no information in the literature dealing specifically with the effect of oxygen on the viability of yeasts and on the rates of fermentation under conditions of rapid fermentation. Both batch and continuous fermentation data (F. F. Pironti, Ph.D. thesis, Cornell University, Ithaca, N.Y., 1971), with an 18% (wt/vol) glucose concentration at 30 C, have shown that air does have a protective effect against product inhibition. By vacuum fermentation, it has also been shown that alcohol is not the only factor contributing to inhibition of growth or fermentation. Substrate inhibition has been shown to have a relatively minor effect compared to the product inhibition at high concentrations of sugar. Pironti's calculations indicated that rates of sugar accumulation intracellularly were higher than the corresponding rate of sugar consumption from batch data. This study was undertaken to determine the effects of controlled oxygen levels, temperature, number of yeast cells inoculated, and addition of sugar in increments rather than in total on the percentage of initial cells inoculated remaining viable during rapid fermentation. MATERIALS AND METHODS Yeast strain. All experiments were conducted with a beer strain of S. cerevisiae kindly supplied by Mario Frati, Genesee Brewing Co., N.Y. The yeast was obtained as a solid paste containing from 3.85 x 109 to 4.04 x 101 cells per g. The yeast was suspended in citrate-phosphate buffer (pH 6.8) and centrifuged to recover the cells used in the experiments. Yeast counts. Based upon the number of yeast cells desired in the inoculated medium, a given weight, generally 1 kg, of yeast paste was slurried with 1 liter of the medium. After thorough dispersion, a 1-ml sample was serially diluted and plated (two plates per dilution) to obtain the initial viable count. The culture medium used for pour plates contained 1.5% maltose, 1.5% malt extract (Difco), and 1.5% agar (Difco). The plates were incubated at 30 C for 48 h, and the final colony count was taken as the average of the two plates for the dilution containing 30 to 300 colonies per plate. Calculations involving cell populations were based upon viable counts. The percentage of viability was determined by dividing the viable count at the desired fermentation time by the initial viable count. Fermentation medium. Clover honey stored at 1 C was diluted with water to provide the 25°Brix sugar substrate. A basal level of 0.25 g of Actiferm per liter (Budde and Westermann, New York, N.Y.), a yeast vitamin mixture, was added to the fermentation medium, and this level is referred to as X. The 0.25 g of Actiferm per liter contained: biotin, 12.5 gg; pyridoxine, 250 jig; meso-inositol, 1.87 mg; calcium pantothenate, 2.5 mg; thiamine, 5 mg; peptone, 25 mg; and ammonium sulfate, 215 mg. The medium 384 APPL. MICROBIOL. S. CEREVISIAE IN RAPID FERMENTATIONS was also supplemented with 1.0 g of (NH4)2SO4, 0.5 g of KPO4, 0.2 g of MgCl2, 0.05 g of NaHSO4, and 5.0 g of citric acid per liter as per formula I of Steinkraus and Morse (11). The basic level of mineral salts-citric acid supplement was referred to as Y. Certain fermentations in this study were conducted using a 2Y level of mineral salt-citric acid supplement and 4X concentration (1 g/liter) of vitamins (Actiferm). The pH of the fermentation medium was adjusted to 4.2, and it was pasteurized at 76.5 C for 30 min and cooled to fermentation temperature before inoculation. Apparatus. (i) Microferm laboratory fermentor model 214 (New Brunswick Scientific Co., New Brunswick, N.J.) was used. A fermentor sampler (New Brunswick model S 21) was used to sample medium during fermentation. (ii) Dissolved oxygen (DO) controller model DO-60 (New Brunswick Co.) was used in conjuction with the above to measure and control the oxygen level. (iii) The fermenting medium was sampled at desired intervals, and cell-free supernatants were obtained for ethanol determinations by centrifuging at 14,000 x g for 10 min in a Sorvall centrifuge model SS-3. All batch fermentations were continued until essentially complete. (iv) A Carle gas chromatograph, model 9000 equipped with a flame-ionization detector, was employed for the separation and quantitation of ethanol. inner diameter) fitted into the instrument to provide on-column injection. The column packing was Poropak Q-S, 100/120 mesh. The column oven was operated isothermally at 150 C. The carrier gas was nitrogen at a flow rate of 20 ml/min. The combustion gases were hydrogen and air, at flow rates of 21 and 300 ml/min, respectively. Sensitivity of the instrument for the analysis was maintained at 2 x 10-9 A full scale. A 2-uliter sample was injected into the instrument with a no. 701 Hamilton syringe. The gas chromatograph was connected to a Hewlett-Packard integrator, model 3370A, to determine the area of the ethanol and internal standard curves. Visual display of the chromatogram was accomplished by means of a Houston "Omniscribe" strip chart recorder with a sensitivity of 1 mV full scale and a chart speed of 1 inch/min (2.54 cm/min). Standard solutions of ethanol were prepared. The standards contained 0.5, 1.0, 1.5, and 2.0% ethanol (wt/vol). A 2% (wt/vol) acetone solution was used as the internal standard. The areas of the peaks were determined by the electronic integrator. The area ratio of the ethanol to the internal standard was calculated as follows: area ratio = area of the ethanol peak/area of internal standard peak. The standard curve was prepared by using the method of least squares. The following equation resulted in: percent ethanol = a + b (peak area ratio of unknown), where a = intercept of standard curve and b = slope of standard curve. Knowing the intercept and the slope, a simple calculation gave the percentage of ethanol in the unknown sample injected. When the ethanol concentrations were high, the samples were diluted 1:5 or 1:10 depending on the concentration, and 1 ml was added to 1 ml of the internal standard. The resulting mixture was analyzed and the percentage of ethanol obtained was multiplied by the dilution factor (L. R. Mattick, Agr. Exp. Station, Geneva, N.Y., and A. C. Rice, Taylor Wine Co., Hammondsport, N.Y., unpublished data). (v) A temperature-compensated refractometer (American Optical Co., Buffalo, N.Y.) was used to determine the percentage of sugar and other soluble solids in degrees Brix. Method of calculating ethanol molecules produced per cell per second. The number of viable cells at different time intervals during the course of the fermentation was determined by serial dilution and plating, as described above, and the results were plotted. Also, the ethanol concentrations, in grams per 100 ml, were determined at the same time intervals by centrifuging the yeast cells and analyzing the ethanol content of the supernatant in the gas chromatograph. The grams of ethanol produced during a specific fermentation time were changed to ethanol molecules by multiplying the moles of ethanol produced times Avogadro's number (6.02 x 1023 molecules per mol). The grams of ethanol produced per 100 ml was plotted against time. By drawing a tangent at the desired point on the curve, it was possible to get the slope or instantaneous rate of ethanol production at that time. Dividing the rate of ethanol molecules produced by the number of viable cells at the same time yielded the number of ethanol molecules being produced per cell per unit of time. RESULTS AND DISCUSSION Effect of DO in the substrate and temperature on yeast viability during rapid fermentation. The percentage of DO in the medium was found to have a considerable influence upon the percentage of cells remaining viable under conditions of rapid fermentation at 30 C. The percentage of yeast cells surviving increased from 2 to 13 to 34 to 60% as the DO content in the substrate was increased from 0 to 13 to 20 to 100% (Fig. 1). The ethanol concentration reached the desired 9.5% (wt/vol; 12% vol/vol) in the substrates containing 0 and 13% DO after 3 h, but reached 8.6 and 7.3% ethanol (wt/vol) in the fermentations in which the DO was maintained at 20 and 100%, respectively, after 5.5 h (Fig. 2). At 15 C, nearly all the yeast cells survived rapid fermentation at 13 and 20% DO concentration in the substrate (Fig. 3), and, at (Fig. 4). These initial studies demonstrated that the lower the fermentation temperature, the better the retention of viability of the yeast. It also was noted that the greater the aeration, the better the retention of cell viability; and, as DO content approached 100% at 15 C, a good rate of cell multiplication was reached with the yeast count nearly doubling after a 6-h fermentation. However, as DO content increased above 13%, the rate of fermentation was inhibited, and fermentation time was extended beyond 6 h, the time arbitrarily set as a maximum for rapid fermentation. The best conditions for rapid fermentation on the basis of the initial studies were fermentation at 15 C and 13% DO, under which 94% of the cells inoculated remained viable after the ethanol concentration reached 9.5% (wt/vol) (6 h). This survival rate would not be acceptable for continuous fermentations in which it is necessary to maintain 100% viability and, preferably, a slight rate of multiplication to offset losses of yeast which must be recovered from the output of the fermentor. Effect of initial cell count on viability during rapid fermentation. To insure that rapid death was not due to the deficiency of any nutrients required for growth or fermentation, the medium was supplemented with twice the usual quantity of ammonium salts, phosphate, and magnesium, along with four times the usual quantity of vitamins generally required in a regular fermentation. The DO was maintained at 13% to insure that oxygen was not limiting. At 15 C, the cells, even at the high initial cell population of 8.3 x 108 cells per ml, showed slight multiplication with the added nutrients (Fig. 6). Thus, the initial cell population by itself was not responsible for the rapid death of cells. The rate of multiplication was progressively increased as the initial cell count was decreased from 1.61 x 108 to 5.9 x 107 to 1.03 x 107 cells per ml at 15 C. However, the fermentations at the two lower levels were no longer rapid. At 30 C, the higher the initial yeast count, the greater the rate of death as the initial cell counts were increased from 1.1 x 107 to 8.0 x 108 cells per ml (Fig. 7). The cells inoculated at the high level of 7.8 x 108 cells per ml died rapidly at 30 C, even though the nutrients and oxygen were not apparently limiting. The loss of the viability of cells inoculated at an intermediate population (1.52 x 108 cells per ml) was not nearly as prominent at 30 C as it was in the fermentation with high cell population. Using an inoculum in the range of 1.1 x 107 cells per ml, it was found that a high level multiplication occurred both at 15 C and 30 C, with the highest maximum population at 15 C (Fig. 6 cells per ml, which was expected to maintain a stable population at 30 C. Viability figures of 2 _/ 100.8% and 99.2% at the end of 1 h and 103% and 98.8% at the end of 4 h were observed for the 7 two respective fermentations (Fig. 8). This 10 confirms the calculations, which were very close demonstrates that, if fermentation time is not of prime significance, conditions of fermentation can be selected which will enable the fermentation to be conducted at 30 C while maintaining a stable yeast population. However rapid fermentations, i.e., a fermentation time less than 6 h, could not be carried out at 30 C with maintenance of yeast viability. The effect of sugar concentration on yeast viability. Adding the 25% sugar in increments of 2.5, 5, or even 15% initially, with 10% sugar added after 1 h, resulted in improved viabilities of 83, 70, and 41%, respectively, compared with 16% viability for anaerobic (0% DO) fermentation started with 250 Brix sugar and continued for 3 h at 30 C, at which time the ethanol content had reached 9.5% (wt/vol; 12% vol/vol; Table 1). The use of 13% DO resulted in a considerable improvement in viability (41 to 79%) at 30 C when the sugar was added 15% at the start with 10% added after 1 h (Table 1). Using an initial concentration of 250 Brix sugar, there was no improvement in viability with an increase of DO from 0 to 13%. With 5% sugar increments, the viability was 70%, even in the absence of oxygen, and it improved moderately (79%) with use of 13% DO. Similarly, the use of 2.5% sugar increments resulted in still higher viability (83%) anaerobically and the use of 13% DO increased viability to 88%. The improvement in viability, with lower levels of sugar added incrementally, can be explained on the basis that the yeast under conditions of aeration at 30 C in a high-sugar medium does not rapidly synthesize its respiratory enzymes due to catabolite repression and, hence, in spite of aeration, has to depend on the glycolytic pathway for its energy requirements. In a rapidly fermenting cell, a further feed-back control on the glycolytic pathway may be expected due to product inhibition. This could cause a depletion of the energy level in the system, which could ultimately bring about a curtailing of the metabolic activity causing death to the cell. By use of incremental feeding, the concentration of sugar in the medium has been maintained at a low level permitting respiratory enzymes to function whenever energy is in short supply. This would lower the rate of ethanol production in the cell and may contribute to the higher percentage of viability maintained during incremental feeding. At 15 C, under anaerobic conditions (0% DO), the addition of sugar in increments of 2.5, 5, or 15% initially, followed by 10% after 2 h, resulted in viabilities of 95, 91, and 80%, respectively, compared with 71% viability for the fermentation started with 250 Brix sugar concentration and continued for 6 h, at which time the ethanol content had reached 9.5% (wt/vol). The improved viability, following the addition of sugar incrementally at 15 C anaerobically, also can be accounted for by a decrease in substrate inhibition. Furthernore, at 15 C a lower rate of metabolic activity can be expected. Thus, the energy requirements also may be much less than at 30 C, and the energy derived from glycolysis could be sufficient to keep the cells alive. At higher concentrations of sugar, substrate inhibition may affect the rate of glycolysis restricting the energy supply for the survival of yeast cells. This deficiency with respect to energy may ultimately cause death to the cell even though the death rate was lower than that observed at 30 C. At 15 C, under aerobic conditions (13% DO), the addition of sugar in increments of 2.5, 5, or 15% initially, followed by 10% after 2 h, resulted in viabilities of 122, 119 and 110%, respectively, compared with 109% viability for the fermentation started with 250 Brix sugar concentration and continued for 6 h, at which time the ethanol content reached 9.5% (wt/vol) ( Table 1). The higher degree of cell multiplication at lower incremental rates may again be due to the influence of oxygen on viability when catabolite repression is minimal at low sugar concentrations. Rates of ethanol produced per cell per second. The rates of ethanol molecules produced in batch fermentations per yeast cell per second, at different temperatures, are presented in Table 2. Rates of ethanol production ranged from about 107 to nearly 3 x 10' molecules of ethanol produced per cell per s. The higher the temperature in the range of 15 to 30 C, the faster the rate of ethanol production. The rates of ethanol production gradually decreased as ethanol concentration increased to 9.5% (wt/ vol; 12% vol/vol). An anomaly was observed in the rates of ethanol produced per cell per second at 30 C. The rate of ethanol production per cell per second appeared to reach its highest point at 2 h, although the highest rate of death was encountered after 30 min at 30 C. We have no explanation of this anomaly at this time. The rates of ethanol molecules produced per cell per second have proven very useful. For example, at 15 C the overall average rate was 4.7 x 107 ethanol molecules produced per cell per s. If the desired ethanol concentration was 9.5% (wt/vol; 12% vol/vol), the yeast cell must produce 9.5 g of ethanol per 100 ml in the time interval specified. The 9.5 g of ethanol is 9.

v3-fos

2016-05-12T22:15:10.714Z

1974-04-15T00:00:00.000Z

8411226

s2

Genetic and phenotypic parameters of body temperature and respiration rate in fayoumi chicks SUMMARY Heritability values for morning and afternoon body temperature and respiration rate were estimated in Fayoumi chicks at hatching and at the ages of 1 , 2 and 3 months. The numbers of chicks used in the four ages were repectively 1424 , 959 , 6 41 and 453. The highest h 2 value of surviving chicks for body temperature was about o.i 5 , which occurred at two months of age, Estimates of h 2 for respiration rate were generally higher than those of body temperature, and for surviving chicks, the highest h 2 value of 0. 25 occurred at i and 2 months of age. INTRODUCTION The fact that different breeds of chicken differ in their body temperature (HILLER M A N and W I I, SO N, I955 ; KA M A R and K H AI, I FA, I g64 ; and I,AMOR!UX and H UTT , 1939 ) and differ also in the response of body temperature to air temperature and relative humidity (Fox, ig5t ;L E E et al., 1945 ;and YEAT!s et al., 1941 ) would suggest genetic differences between breeds with respect to these characters. In fact such differences were used by H U TT and C RAWFORD ( 19 6 0 ) in selecting for high and low body temperatures. Respiration rate is also a character which has its significant role in the heat tolerance mechanism and the stabilization of body temperature in birds. This work is the first part of some genetic studies on heat tolerance in Fayoumi, a native breed of chicken. It deals with the heritabilities of body temperature and respiration rate of chicks up to three months of age, together with body weight, a production characteristic. MATERIAL AND METHODS Out of five batches of chicks hatching during December and January, a total number of 1424 unsexed one-day-old Fayoumi chicks was used for this study. Chicks were reared in floor brooders till three months of age under the normal managerial conditions of the Poultry Breeding Farm, Faculty of Agriculture, Cairo University. At hatching and at each of the three ages 1 , 2 and 3 months chicks were weighed and rectal temperature (in OF) and respiration rate were measured twice, once early in the morning (between 6 and 8 o'clock) and once in the afternoon (between 3 and 5 o'clock). Respiration rate per minute was taken as double the actual count measured twice in 30 seconds. Symbols used for traits are : t and T for morning and afternoon body temperatures respectively, r and R for the corresponding respiration rates and w for body weight ; with subscripts o, 1 , 2 and 3 denoting age in months. The following Table i presents the numbers of sires, dams and offspring used in the four ages studied together with the coefficients of the sire and dam components of variance. It will be noted that the number of chicks measured decreased drastically by age. This is not due to a very high mortality rate, which, however, reached the value of 23 p. 100 by the end of the experiment. But the numbers measured were those left, for this particular experiment, randomly chosen from the survivals at any particular age. However, the term « survivals » will be used to denote chicks measured at older ages. Data were corrected for hatch effect before performing the ordinary hierarchical analyses of variance. Genetic variances (a2 were taken as double the sum of the sire and the dam components of variance. Heritabilities (h 2 ) were then taken as a./af, ; ap being the phenotypic variance. Heritability values (beside the other different parameters) for traits measured at earlier ages, were again recalculated using the earlier performances of survivals to later ages. Thus, for instance, h 2 for to had four estimates using the performances of the four survival groups, while h 2 for t 1 had only three estimates. A second subscript (A, B, C and D) will be added to denote the survival group ; T 2D will be the afternoon body temperature (T) at the second month of age ( 2 ) of the 453 chicks that « survived » till three months of age (survival group D). Comparing the hatching weight (w a ) of the four survival groups, it can be concluded that the three groups B, C and D represent random samples of the original group A with respect to this character, since all have almost the same mean. This conclusion is supported by the fact that the genetic (and phenotypic) variances showed almost the same magnitude in the four groups of chicks. A similar conclusion could in fact be drawn with respect to w when we compare means only, but the amount of genetic and phenotypic variances would not support this conclusion. For there it is clear that the groups which survived till the second and the third month of age, both, had-beside slightly higher means-higher genetic and lower phenotypic variability. But with regard to ? ' 2 it can be seen that the means, se and a ; , are not much different in w,c and W ID . With respect to the heritability values, very high estimates are obtained for w a in the four survival groups (in the order of o.8). This is not unexpected and is explained usually by the presence of maternal effects. Other estimates of h 2 of body weight at different ages are not different from those obtained on the Fayoumi (see Ezzeldin, 1970 ). Characters w lc and WID showed higher heritability values compared to w lB , as would be expected from the previous discussion. II. -Body temperature In comparing the morning body temperature (to) of one-day-old chicks (which would give the picture of the basal condition of the body before the day activity), small increases can be observed between the means of the four survival groups (table 3 a). This is connected with a gradual decrease in phenotypic variance and a very marked increase in genetic variance. The amount of genetic variance, however, is very small indeed, being 0 . 055 in group A and 0 . 203 in group D. The heritability values of this character, therefore, were higher in groups surviving to older ages, reaching the maximum value of o.i8i in to D . A similar picture can also be drawn with respect to the afternoon body temperature of the one-day-old chicks (To). However, the values of G i and a g of To were always lower than the comparable values of t a in the four survival groups, and the heritability values of To are all lower than the corresponding values of to of the same survival groups. It may be also noted that, apart from the two equal mean values of t s n and T 3D , all the T means are higher than the corresponding t values measured on the same chicks the same morning. The morning body temperature of the one-month-old chicks (t i ) showed almost the same mean for the three groups of survivals, which was markedly higher than the comparable to. It is clear that morning body temperature increased about 2 . 5 5 °F after one month from hatching, and stayed constant thereafter at about io6.8-F. It seems that this rise in body temperature occurs earlier, at about 4 -10 days after hatching (FREEMAN, 1971 ;L AMOREUX and H U TT, 1939 ). The genetic and phenotypic variances, however, showed different trends. The phenotypic variance crp of the morning body temperature increased drastically from hatch to one month of age. Its value was about z. 5 for t o n and increased to more than 34 in t lB . This observation may be readily explained by the fact that chicks at hatching are all still poikilotherms, influenced highly by the incubator and the brooder temperatures, and thus showing small amounts of observed variance in body temperature (to and To) between chicks. However, another sudden increase in phenotypic variance occurred also at three months of age (a 2 p of t aD and T 3 n were more than 100 ). One may guess that the brooder temperature imposed still some restrictions on phenotypic variability in body temperature between individuals in the earlier ages of one and two months. At the age of three months, however, chicks were better feathered and such restrictions are expected then to be much less. The genetic variance of t showed negative values in the two cases t 1B and tg n . Considerable ag values occurred in two cases only ; namely t lc and t zo . The heritability in these last two cases reached the values of 0 . 13 6 and 0 . 145 , respectively. It seems then that the only significant heritability value with respect to morning variance ( G $ was 5 9 . 4 in ron and 54 . 2 in y o n) and still by a marked decrease in genetic variance (a2, was 7 . 3 in r(,Aand 2 .q in r oD ). This resulted in the decrease of heritability values from the significant value of o.i 4 2 in r oA to the insignificant value of 0 . 045 in y o n. The means of R o showed lower values than their corresponding r o values, though the differences are of very small magnitudes, but the phenotypic variances of R o were markedly higher. The genetic variance was equal to about 12 in R o n (resulting in the highly significant heritability of o.16 4 ) and decreased drastically in the groups surviving to older ages till it showed the negative value of -1 . 9 in R OD . The means of rand R decreased by the advancement of age, and also, after the initial y o and R o values, the R means showed higher values than those of r measured on the same chicks the same day. The highest amount of decrease occurred in both r and R between one and two months of age. The role of respiration becomes less in regulating body temperature as the chick develops more feathers, since feathers increase body insulation. Phenotypic variance, too, decreased gradually by the advancement of age, showing the biggest decrease at one month of age ; values for R being always higher than the comparable r values till both reached almost the same value at three months of age. The genetic variances of r l , which showed higher values compared to those of r o , showed a tendency to increase in groups surviving to older ages. The genetic variance of R IB was a little bit lower than that of R o n, but the tendency of groups surviving to older ages to have higher aa is also clear. Thus at the age of one month all h 2 values of r l and R l were of high and considerable magnitudes, those of r. being always higher than the comparable R l values. At two months of age, the heritability value was o.245 for y 2 c and o.z 4 8 for R a c, and at the third month the estimate of heritability decreased to 0 . 15 8 in r3 D and to o.i22 in R zD .

v3-fos

2018-04-03T05:22:52.403Z

1974-06-01T00:00:00.000Z

42567530

s2

Combined effects of water activity, solute, and temperature on the growth of Vibrio parahaemolyticus. Vibrio parahaemolyticus was grown at 36 C in tryptic soy broth (pH 7.8) containing added levels of NaCl ranging from 0.5 to 7.9% (wt/wt). The fastest generation time was 16.4 min in tryptic soy broth containing 2.9% NaCl (TSBS) which corresponded to a water activity (a(w)) of 0.992 (+/-0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower a(w), respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a(w) to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a(w) with glycerol was less inhibitory to growth than similar a(w) reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a(w) levels lower than 0.992. Minimal a(w) for growth at 15, 21, 29, and 36 +/- 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a(w) was generally most tolerable at 29 C, whereas higher minimal a(w) for growth was required at 15 C. Solutes added to TSBS to achieve reduction in a(w), minimal a(w) for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a(w) for growth of V. parahaemolyticus depends upon the solute used to control a(w). which corresponded to a water activity (a,) of 0.992 ( 0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower aw, respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a, to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a, with glycerol was less inhibitory to growth than similar a. reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a, levels lower than 0.992. Minimal a, for growth at 15, 21, 29, and 36 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a, was generally most tolerable at 29 C, whereas higher minimal a, for growth was required at 15 C. Solutes added to TSBS to achieve reduction in aw, minimal a, for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a, for growth of V. parahaemolyticus depends upon the solute used to control aw. The relationships between available water content and the potential for spoilage of foods by microorganisms have been of interest for many years. Various minimal, optimal, and maximal moisture levels, usually expressed in terms of water activity (aj), have been reviewed (17,20). Much of the available data on a, requirements for bacteria have resulted from studies on foodborne pathogens such as salmonellae (3,4,6), staphylococci (5,15,16,19), and Clostridium spp. (1,7,10,18). In light of the recent recognition of Vibrio parahaemolyticus as a cause of foodborne disease outbreaks in the United States (2) and the lack of definitive information regarding the organism's tolerance to reduced a, levels, several experiments were designed to determine the growth response of V. parahaemolyticus over a wide range of a,. In the present study, the optimal a, in a medium containing added NaCl was established. Effects from minor changes in optimal a, levels resulting from the addition of several solutes to growth media on lag times of V. parahaemolyticus are discussed. Minimal a, levels for growth at 15,21,29, and 36 C were achieved by the addition of solutes to a basal medium. MATERIALS Media. The basal medium used for all investigations was tryptic soy broth (TSB, Difco), which contains 0.5% NaCl when prepared according to the manufacturer's directions. For studies involving growth of the organism at elevated NaCl levels, quantities of NaCl were added to known volumes of TSB, and the percentage of NaCl was expressed on the basis of grams of NaCl per grams of final NaCl-TSB mixture, assuming the density of TSB to be 1.0. Each of the remaining solutes studied (KCI, glucose, sucrose, glycerol, and propylene glycol) were added individually to TSB containing 2.93% NaCl (TSBS) in progressively increasing amounts to achieve a. values above and below which V. parahaemolyticus would grow. Percentages of these solutes, which are referred to throughout this report, were calculated as grams of solute per grams of final mixture (solute plus TSBS), again assuming the density of TSBS to be near 1.0. All growth media were adjusted to pH 7.8 by adding 2 N NaOH, dispensed into either 13 by 100 mm or 16 by 150 mm screw-cap tubes and sterilized at 121 C for 15 min. Random measurement of pH after sterilization revealed changes of no greater than +0.2. Growth studies. Since V. parahaemolyticus is facultatively halophilic, an initial experiment was designed to determine the optimal NaCl concentration at which the organism would grow. Strain M5250J-2 was cultured in TSBS at 29 C on a gyratory shaker (150 rpm) for 16 h. The culture was diluted 100-fold in distilled water containing 0.1% peptone (Difco) and 3.0% NaCl; 1 ml of the diluted culture was then inoculated into 150-ml portions of fresh TSB containing concentrations of NaCl ranging to 7.87%. The 500-ml Erlenmeyer flasks containing the cultures were returned to the shaker at 29 C. Samples were withdrawn at selected times and appropriate dilutions were made in the peptone-NaCl diluent prior to surface-plating on TSBS containing 1.5% agar (TSBSA) and on thiosulfate citrate bile salts sucrose agar (TCBS). Counts were made after 12 h (TSBSA) and 24 h (TCBS) of incubation at 36 C, and generation times were calculated for the organism cultured in each of the TSB media containing added NaCl. The above experiment showed strain M5250J-2 to have the fastest generation time in TSB containing 2.93% NaCl (TSBS). Addition of various quantities of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol to TSBS was made to achieve only slight reductions in a, levels. The effects of these a. reductions on lag times of strain M5250J-2 were then examined by measuring absorbance of the growing cultures at 620 nm after inoculation with a loop of 16-h TSBS culture. Growth temperature for the inoculum and the test cultures was 36 C. Amounts of solutes added, their weight percents, and resulting a. are summarized in Table 1. Minimal a, for growth of each of four strains of V. parahaemolyticus at 15, 21, 29, and 36 C were determined. Each strain was cultured in TSBS for 16 h at 29 C and standard loop inocula were transferred to 10-ml portions of TSBS containing individually added quantities of test solutes. Each test was performed in quintuplicate. In all cases, sufficient solute was added to achieve several a, levels both above and below that required for growth. Caps were tightened on the tubes (16 by 150 mm), and the inoculated media were incubated for 20 days in walk-in incubators adjusted to 15,21,29, and 36 ± 0.2 C. Obvious turbidity during the 20-day period was recorded as positive growth, and tubes were discarded. After 20 days all remaining tubes were examined for number of viable cells by plating on TCBS and for number of total cells by using a Petroff-Hauser counting chamber. Tubes were recorded as negative if viable cell population and direct microscope counts per milliliter were both less than the original number of cells per milliliter of culture at the time of inoculation. Direct counts were necessary to determine whether cell division was followed by death of significant numbers of cells during the 20-day incubation period. Determination of aw. Equilibrium relative humidity measurements were made at 29 C with Hygrosensor elements (no. 4-4822; Hygrodynamics, Inc., Silver Spring, Md.) mounted in lids of 8-oz jars. Hygrometer sensors were attached to a Hygrometer Indicator (model no. 4-4900; Hygrodynamics, Inc.) and an equilibration time of not less than 8 h was allowed before measurements were recorded from 25-ml portions of each test solution. Sensory calibrations were made against saturated KNO.. Mean values were determined from triplicate readings for several concentrations of each solute and sorption isotherms were plotted (Fig. 1). All aw levels reported in this paper were taken from isotherm curves. Accuracy of the measuring system is considered to be no better than ±0.005 aw. However, for purposes of comparison, as will become evident upon examination of results, data are presented at the 40.001 aw level. RESULTS AND DISCUSSION Inhibitory effects of aw levels higher and lower than optimum on the growth and metabolism of several bacterial species have been reported (3,5,6,16,17). Extreme sensitivity to relatively minor variations in osmotic and ionic conditions in growth media have been most dramatic with V. costicolus (8) and V. metchnikovi (13). Reduction in aw from 0.999 to 0.995 resulted in a fivefold increase in the rate of growth of V. metchnikovi. Further reduction in aw generally resulted in decreased rates of growth, depending upon the solute added to control aw and the nutrient composition of the growth medium. Several reports indicate conflicting ranges and optima for percentage of NaCl tolerance for V. parahaemolyticus (11,21,22, reviewed by 14; C. R. Lazarus and J. A. Koburger, Abst. Southeastern Branch Amer. Soc. Microbiol., 51st and 52nd Ann. Mtg., p. 19,1973). It is difficult to compare these data because methods for preparing "percent NaCl" levels in media were not detailed and aw levels were not reported. Initial experiments were therefore designed to determine the optimal NaCl concentration (and corresponding aw) for growth of V. parahaemolyticus by using TSB as a basal medium. Results are shown in Fig. 2. As aw was decreased from 0.998 to 0.992 (0.5 and 2.93% NaCl, respectively), the generation time of V. Fig. 3; levels were calculated from sorption isotherms shown in Fig. 1. parahaemolyticus M5250J-2 decreased from 24.4 to 16.4 min. Increased NaCl levels above 2.93% prolonged generation times. Whether aw manipulation by the addition of other electrolytes or nonelectrolytes alone or in combination to TSB would have resulted in different aw optima and faster growth rates was not determined. The 16.4-min generation time was considered to result from nearly optimal culture conditions, and therefore TSBS (TSB containing 2.93% NaCl) was used as a basal medium in subsequent studies involving the effects of added solutes on lag phase extension and minimal aw tolerance of the four test strains. Realizing that only slight differences in aw achieved from the addition of NaCl to the growth medium resulted in substantial differences in generation times of strain M5250J-2, it was decided to measure the effects of low amounts of added KCl, glucose, sucrose, glycerol, and propylene glycol, in addition to NaCl, on lag times of the organism. Data are plotted in Fig. 3. Grams of solute added per 100 ml of TSB or TSBS, calculated solute concentrations, and corresponding aw levels are summarized in Table 1. Some trends can be observed in these data. NaCl studies show that 0.992 a, results in the shortest lag time, fastest growth rate, and highest total biomass production. Departure from 0.992 a, resulted in longer lag times with slower growth rates and depressed cell production. These data tend to confirm those derived from generation time studies. Addition of solutes to TSBS (0.992 a.) reduced a. in all cases, but the magnitude of change in growth parameters was varied, depending upon the solute added. Glycerol had the least inhibitory effect on V. parahaemolyticus of all solutes tested. Addition of glycerol to achieve a 0.961 a, Table 1 for summary of media preparation. extended the lag phase only by 4 h compared to greater lag phase prolongation at even higher a. levels for the other test solutes. Inhibitory effects of NaCl and KCl were similar at comparable reduction in a, levels, whereas propylene glycol, glucose, and sucrose, in that general order, were most effective in delaying logarithmic growth and depressing cell production by strain M5250J-2. The glycerol data are in agreement with those reported for Staphylococcus aureus survival (15) and enterotoxin production (19) and growth of Salmonella oranienburg (4), Bacillus cereus (9), and Clostridium perfringens (10), wherein glycerol was found to be less inhibitory than other test solutes. Marshall et al. (12), on the other hand, reported that at aw levels between 0.96 and 0.90 the inhibition of S. aureus growth rate was about 10% greater in glycerol than in NaCl. Glucose and propylene glycol proved to be most bacteriostatic to V. parahaemolyticus when compared to the other solutes at similar aw levels. Total inhibition of growth of V. metchnikovi in nutrient broth containing glucose in amounts to achieve aw less than 0.997 has been noted (13). These authors reported no growth of the organism in brain heart infusion broth containing glucose. Glucose appears, therefore, to exhibit a toxic effect on V. metchnikovi and V. parahaemolyticus. By-products arising from chemical reactions between glucose and medium constituents during sterilization may account for these inhibitors. The reasons for V. parahaemolyticus sensitivity to propylene glycol cannot be explained. Table 2 shows the minimum a, for growth of V. parahaemolyticus in TSB and TSBS adjusted to reduced a, levels by the addition of various solutes. Calculated concentrations of added solutes are also listed. The four test strains responded similarly in their minimal a, levels at various temperatures. In most cases the a, levels listed in Table 2 are representative of at least three of the four strains. Therefore, only one minimal a, is listed for each solutetemperature combination. In general, solutes which were more inhibitory with respect to prolonging lag phase of growth were also more effective in completely inhibiting growth. Glycerol was least effective in controlling growth, followed by NaCl and KCl which were approximately equal, and then sucrose, glucose, and propylene glycol. The 29 C incubation temperature proved to be most satisfactory for the organism's tolerance to low aw, whereas 15 C adversely effected the response to aw stress. V. metchnikovi was reported to have lower tolerance to glycerol than to NaCl when aw adjustment was made in quarter-strength brain heart (12). Although the source of broth was not stated by the authors, if a Difco or BBL product was used, the initial concentration of NaCl was 0.5%. In view of the relatively high ionic strength required in growth media by Vibrio spp., the reversal in apparent tolerance of V. metchnikovi and V. parahaemolyticus to NaCl and glycerol might partially be explained on the basis of electrolyte concentration of the basal media used in the two experiments. The TSBS basal medium used to establish solute tolerances in the present study may have provided sufficient electrolyte to satisfy V. parahaemolyticus requirements. Therefore, addition of a nonelectrolyte such as glycerol to TSBS may have resulted in minimal a, levels which measured tolerance to the nonelectrolyte instead of tolerance to a combination of stresses induced from low electrolyte and high nonelectrolyte concentrations concurrently. The relationship between limiting a, levels for growth of microorganisms and the solute added to achieve those levels is unclear. Several authors (7,17) have stated that biological response to a, by some organisms is independent of the types of solutes used to reduce the aw. Other reports have shown that nutrient availability (3), pH (1), oxygen level (6,16), and moisture content (15), in addition to the test solute, effect a microorganism's ability to grow at limiting aw. It appears that the limiting aw for growth of the four V. parahaemolyticus strains examined in this study depends upon the solutes used to attain these lower limits. Notwithstanding the possibility of nutrient dilution effects inherent in the methods employed to prepare the test media, the differences in physico-chemical properties of the solutes apparently have a significant influence on the ability of V. parahaemolyticus to tolerate suboptimal aw levels. Further experiments are required to determine the response of V. parahaemolyticus to low aw levels achieved by the addition of other halogen salts to growth media. Studies involving the combined effects of various electrolytes and non-electrolytes on tolerance of the organism at reduced aw would also provide valuable information on growth characteristics of this facultative halophile. ACKNOWLEDGMENT The technical assistance of B. Vaughn is gratefully acknowledged.

v3-fos

2020-12-10T09:04:12.333Z

1974-04-01T00:00:00.000Z

237230676

s2

Survival of Vibrio parahaemolyticus in Cooked Seafood at Refrigeration Temperatures The growth and survival of two strains of Vibrio parahaemolyticus isolated during food-borne gastroenteritis outbreaks in Japan and surface inoculated on cooked shrimp, shrimp with sauce, or cooked crab were tested at various refrigeration temperatures during a 48-h holding period. On cooked shrimp and crab, the vibrios grew well at 18.3 C, but their numbers declined gradually at 10 C and below. At 12.8 C, vibrios remained static for the most part. Thus, it appeared that 12.8 C was the borderline temperature for growth of the organism on cooked seafood. When cocktail sauce was added to surface-inoculated shrimp at a ratio of 2:1, the vibrio die-off rate was accelerated. In the shrimp and sauce few cells remained after 48 h, but in the sauce alone die-off was complete at 6 h. The growth and survival of two strains of Vibrio parahaemolyticus isolated during food-borne gastroenteritis outbreaks in Japan and surface inoculated on cooked shrimp, shrimp with sauce, or cooked crab were tested at various refrigeration temperatures during a 48-h holding period. On cooked shrimp and crab, the vibrios grew well at 18.3 C, but their numbers declined gradually at 10 C and below. At 12.8 C, vibrios remained static for the most part. Thus, it appeared that 12.8 C was the borderline temperature for growth of the organism on cooked seafood. When cocktail sauce was added to surface-inoculated shrimp at a ratio of 2: 1, the vibrio die-off rate was accelerated. In the shrimp and sauce few cells remained after 48 h, but in the sauce alone die-off was complete at 6 h. The facultative halophile, Vibrio parahaemolyticus, is recognized as a public health hazard in seafoods of United States and foreign origin (13). Recent outbreaks of food poisoning in the United States were associated with the consumption of crab or shrimp contaminated with this organism (4)(5)(6)(7). Foods contaminated with V. parahaemolyticus through inadequate preparation or handling may be held under conditions that favor the growth of bacteria and increase the risk to the consumer. Generation times as low as 12 min were observed when these organisms were inoculated into sea fish (11). Minimal temperatures reported for V. parahaemolyticus multiplication were 5 C (3) and 8 C (1) in artificial media, and 10 C in oyster homogenate (15) and the marine environment (10). Under favorable growth temperatures, extensive multiplication may occur that probably increases the risk of food-borne illness. Restaurants and cafeterias commonly prepare seafood salads in advance of serving time and store them in the refrigerator until they are displayed on the serving line. Temperatures as high as 12.8 C in refrigerated showcases (2), 10 C in domestic refrigerators (19), and 15.6 C in coolers used in the blue crab industry (12) have been recorded. Such deviations from optimum refrigerator temperatures might well permit multiplication of contaminating V. parahaemolyticus. A few studies (8,9,15,18) of the survival of V. parahaemolyticus inoculated into the tissues or into homogenates of shrimp and oysters have been conducted. But such studies have limited applicability to the hazards of storing poorly refrigerated, contaminated seafood. This study was therefore undertaken to determine the effects of storage temperatures representative of both good and poor refrigeration on the growth and survival of V. parahaemolyticus on the surface of cooked seafoods such as shrimp and crab. MATERIALS AND METHODS Cultural methods. The two cultures of V. parahaemolyticus used in this study were Yanagisawa, 04: K9 (obtained from H. Zen-Yoji, Tokyo Metropolitan Research Laboratory of Public Health, Tokyo) and 9382, 04: K11 (obtained from Y. Miyamoto, Kanagawa Prefectural Public Health Laboratory, Yokohama). Both cultures were isolated during foodborne gastroenteritis outbreaks in Japan. The media and culture conditions used for maintenance of stock cultures as well as for verification of strains have been previously described (16). Both strains exhibited properties that have been well established for identifying pathogenic V. parahaemolyticus (14). Kanagawa hemolysis was determined on Wagatsuma agar (Eiken) that contained 10% washed human red cells. Serotyping was accomplished by slide agglutination with 8 monovalent 0, 8 polyvalent K, and 52 monovalent K antisera (Toshiba Kazaku Co., Ltd., Tokyo). Inocula for a given experiment were prepared from strains grown on Trypticase soy agar (BBL) containing 3% NaCl at 35 C for 18 to 22 h. Cells were washed twice and resuspended in buffered physiological saline to a known optical density. Preparation of seafood. Frozen whole jumbo shrimp in shells and frozen pasteurized Alaskan king crab meat were used in this study. Samples were cooked at 100 C for 6 min; the cooking water was then 657 poured off, and cold, sterile distilled water was added. Shells were aseptically removed from the shrimp. Twenty-five grams of either crab or shrimp was added to sterile, 3-oz (88.69-ml) plastic bottles. Samples were equilibrated at 35 i 1 C and surface inoculated with 1 ml of cell suspension adjusted to provide initial concentrations of approximately 104 organisms/g. Bottles were sealed in plastic bags and immersed in water baths set at the desired test temperatures. Duplicate plates were prepared from appropriate dilutions of duplicate samples in modified Twedt medium (17) at 0, 6, 12, 24, 36, and 48 h. Plates were incubated at 35 C for 42 to 44 h. V. parahaemolyticus were never detected in uninoculated control samples plated at 0 and 48 h. In certain assays, 50 g of a commercial sauce (Seafood Cocktail Sauce, Crosse & Blackwell Co., White Plains, N.Y.) was added to 25 g of inoculated shrimp to approximate a commercial seafood cocktail that is produced and then frozen for retail sale. In other assays, samples of sauce were inoculated and tested for survival. APPL. MICROBIOL. Verification. Five colonies from each of four countable plates at each incubation temperature in every experiment were picked to test in Trypticase soysalt broth for verification of V. parahaemolyticus. The biochemical tests performed on each included growth in 0, 1, 8, and 10% NaCl; production of acetoin, indole, and H2S; and fermentation of glucose and sucrose. Serotyping was performed by slide agglutination. RESULTS The growth and survival of two strains of V. parahaemolyticus that were surface inoculated on whole shrimp held at six refrigeration temperatures are shown in Table 1. The vibrios grew well at 18.3 C, but their numbers declined from 0.5 to 1 log at 10 C and below during the 48-h holding period. At 12.8 C, strain 9382 declined 1 log, and strain Yanagisawa remained relatively static. Table 2 illustrates the effect of added cocktail sauce on the growth and survival of vibrios on shrimp. In most cases counts fell 9382). In sauce alone, few viable cells were more than 1 log in 24 h at all temperatures. Few present at 6 h, and die-off was complete at 24 h organisms remained at 48 h. The rate of decline at all temperatures. The growth and survival of seemed less at 18.3 C than at the other tempera-V. parahaemolyticus on cooked crabmeat were tures. Indeed, multiplication occurred after 36 h similar to those observed on shrimp (Table 3). at this temperature in one experiment (strain The organisms grew well at 18.3 C, remained static (except for the small increase in experiment 1 at 48 h) at 12.8 C, and declined approximately 0.5 log at 7.2 C and 1 to 2 logs at 1.6 C during the 48-h holding period. DISCUSSION Our results indicate that V. parahaemolyticus inoculated onto the surface of cooked shrimp or crab gradually decline in numbers at incubation temperatures of 10 C and below and multiply if held at 18.3 C. Apparently, 12.8 C is the borderline temperature for growth of this organism on cooked seafood. The Yanagisawa strain remained nearly static, and the 9382 strain declined 1 log during the 48-h storage period. By utilizing different experimental menstrua and methods, other investigators have obtained analogous results. Vanderzant and Nickelson (18) observed a decrease of 2 logs during the first 2 days of storage at 3, 7, and 10 C of approximately 105 V. parahaemolyticus cells injected into whole uncooked shrimp. In shrimp homogenates inoculated with approximately 5 x 104 cells/ml, results were quite different. An initial slight increase in numbers over the first 12 h at all three temperatures was followed by a gradual decrease. Johnson and Liston (8) reported a slow decline in numbers of a V. parahaemolyticus strain after 2.5 days of storage at 11 C and below in depurated oysters naturally contaminated with 5.8 x 104 cells/g. Thomson and Thacker (15) inoculated V. parahaemolyticus strains into oyster homogenates at an initial level of approximately 5 x 103 cells/ml. They observed multiplication at 10 C and above and no change at 8 C, with a gradual decline of 1 to 2 logs at 4 C and below during 1 week of storage. Johnson et al. (9) reported little or no apparent decrease of V. parahaemolyticus in naturally contaminated oyster shellstock stored for 3 weeks at 4 C. Reports in the literature demonstrate that domestic and commercial refrigerators exhibiting fluctuating temperatures capable of supporting Vibrio survival or multiplication on cooked seafood are not uncommon. Bauman (2) reported refrigerator showcases to cycle between 4.4 and 12.8 C during a 12-h period. When van Walbeek et al. (19) tested domestic refrigerators in the early morning hours to avoid fluctuation caused by frequent opening, they recorded temperatures as high as 10 C. Commercial coolers exhibited temperatures up to 15.6 C during a survey of the blue crab industry by Phillips and Peeler (12). When commercial sauce was added to sur-face-inoculated shrimp, Vibrio decline was accelerated. In most cases, the die-off rate was greater than 1 log in 24 h. Only minimal numbers were present at 48 h. The explanation for this rapid decline may lie with the acidity of the sauce (pH 3.3 to 3.4). In sauce alone, Vibrio die-off was virtually complete in only 6 h. Since the shrimp was surface inoculated before admixing with sauce, it is very likely that the bacteriocidal effects of the acid pH were modified by buffering that would result from the association of vibrios with shrimp tissue. The gradual decline of V. parahaemolyticus on the surface of cooked seafood at refrigerator storage temperatures of 10 C and below can hardly be considered to eliminate the public health hazard inherent in a contaminated product. The danger of gastroenteritis is still present for the consumer, either from massive numbers of V. parahaemolyticus or from modest numbers of a highly infectious strain.

v3-fos

2020-12-10T09:04:12.639Z

1974-04-01T00:00:00.000Z

237233233

s2

Isolation of Edwardsiella tarda from Swine Edwardsiella tarda was isolated from the intestinal tract of a 2-month-old pig. This is the first reported isolation of Edwardsiella tarda from swine in the United States. Swine have been reported as potential carriers of Edwardsiella tarda, but pathogenicity of this organism for swine has not been determined. Although the pig had access to several farm ponds, the exact source of infection was not determined. the exact source of infection was not Edwardsiella tarda was recognized in the United States as early as 1959, but it was not until 1965 that it was proposed as a new genus and species in the family Enterobacteriaceae (5). At that time, 37 E. tarda isolants had been reported. These isolants were from the United States and several other countries. Two of these isolants were from animal sources, 34 were from humans, and 1 source was unidentified. One of the animal isolants was from a bovine with diarrhea, but the source of the other was not indicated (4). E. tarda was first reported in Japan in 1962 and was designated the "Asakusa Group" (9). It had been recognized in Japan since 1959. In Japan, of the 256 cultures which were originally studied, 248 were isolants from snakes, 2 were from seals, and 5 originated from the feces of humans affected with symptoms of gastroenteritis. Isolation of the organism in man has been reported from feces, wounds, blood, and urine (2,5,7,9). It has also been incriminated as the etiological agent of a case of fatal meningitis (10). In the United States, E. tarda has been reported from animals in zoological surroundings and in aquatic environments-E. tarda has been recovered from a sea lion, two alligators, and an Australian skink (11,12). It has also been reported to cause diarrhea and severe enteritis in an ostrich (12). It has been isolated from several species of turtles in the Southeastern and Southern United States (6). It has also been recovered from the common snapping turtle (Chelydra serpentina) in central Missouri. Most recently, E. tarda has been reported as a pathogen of channel catfish (Ictalurus punctatus) (8). There have been two reports of E. tarda isolation from swine. The first isolation was from pig bile which was collected from apparently healthy animals in Philippine abbatoirs (1). The second is reported from an animal in Vietnam. Our isolation of E. tarda from a pig in Missouri apparently constitutes the first finding of E. tarda in swine in the United States. MATERIALS AND METHODS On 31 July 1970, a 2-month-old pig was presented to the Veterinary Medical Diagnostic Laboratory, School of Veterinary Medicine, University of Missouri, Columbia, Mo., from a swine herd producer in Williamsburg, Mo. A necropsy was performed on the animal immediately after it was killed. Tissue specimens were removed aseptically from several organs and submitted for bacteriological examination. Their surfaces were seared with a hot spatula, and a sample was removed with sterile scissors and forceps and inoculated onto several agars. Trypticase soy agar enriched with 5% sheep blood and MacConkey agar were utilized for culturing all tissue. In addition, brilliant green agar was utilized when the small and large intestines were cultured. All media were products of BBL, Cockeysville, Md. At the time the bacteriological sample was taken, a duplicate tissue specimen was collected from select organs and placed into 10% buffered Formalin for histopathological examination. Histopathological examination was performed on slides stained with hematoxylin and eosin. RESULTS The pig was one of eight in a litter and had been weak from birth. Its litter mates were healthy and showed no signs of disease. Rectal temperature prior to death was 101 F. Before death, the animal went through a period of diarrhea and depression and did not respond to medication. Postmortem examination failed to reveal remarkable lesions in the abdominal and thoracic cavities. The stomach and small intestine were empty, the large intestine contained a semi-formed stool, and the spleen was pulpy. The heart was very flabby and had serous atrophy of fat depots. There were no remarkable changes in the lungs and endocrine glands. Histopathological examination of the kidney revealed petechiae and congestion in the area of collecting tubules, exhaustion of lymphoid nodules in the spleen, infiltration of leukocytes along portal triads of the liver, and diffuse pneumonitis. No other remarkable histological changes were observed. Microbiological examination of the lung and liver failed to reveal bacterial growth after 48 h. Culture of the small intestine resulted in a scant growth of Escherichia coli within 24 h. E. tarda and E. coli were isolated from the large intestine in heavy concentration within 24 h. The organisms were identified by the methods of Edwards and Ewing (3). Identification of our isolant was confirmed by the Center for Disease Control, Atlanta, Ga. DISCUSSION At the present time, little is known about the pathogenicity of E. tarda for animals. It has been associated with disease in the ostrich (12), in channel catfish (8), and found in association with bovine diarrhea in one case (5). Although E. tarda has been isolated from swine in two countries, no specific lesions have been reported. The only lesion observed in this case was that of infiltrating leukocytes along the portal triads and diffuse pneumonitis. Both of these lesions are common in infectious diseases and septicemias and cannot be considered as being specific lesions associated with E. tarda. Since the necropsy was performed shortly after death, it was felt that bacterial transmigration had not occurred and that the organisms recovered were a true picture of the intestinal bacterial flora at the time of death. The E. coli which was recovered from the small intestine was not considered to be a pathogen because of the small number present. Experience has shown that if E. coli is a significant etiological factor, large numbers of the organisms will be present in the upper intestine which was not the case here. Biochemically, the organism is similar to Salmonella spp. and, perhaps in the past, the organism might have been falsely identified as a Salmonella spp. It is easily differentiated from salmonellae in that it is indole positive and does not ferment mannitol. Its serological and bio-chemical properties have previously been reported (4). Retrospectively, we learned that the swine herd from which the pig came had access to several farm ponds. E. tarda is present in many amphibians and snakes (6,9) but has not been associated with disease. It is interesting to speculate that reptiles or amphibians may act as reservoirs of infection for domestic animals. One report indicated swine may act as carriers of E. tarda as the organism localizes in the gall bladder (1). If this is true, swine may then shed the organism which would return to the ponds and contaminate other reptiles and amphibians. To determine the importance of this organism for swine and other animals, it is essential that diagnostic laboratories be alerted to the possibility that E. tarda may be carried by swine. It is also important to recognize this species and not to confuse it with other enterobacteriaceae. At the present time, it remains for the organism to be incriminated as an enteric pathogen of swine.

v3-fos

2019-03-19T13:12:33.504Z

1974-06-01T00:00:00.000Z

237145733

s2

Plaque Assay for Avirulent (Lentogenic) Strains of Newcastle Disease Virus Avirulent (lentogenic) strains of Newcastle disease virus form plaques on chicken embryo lung monolayers in 48 to 72 h. Newcastle disease virus (NDV) strains can be divided into three categories on the basis of virulence: lentogenic (avirulent), mesogenic, and velogenic (fully virulent) groups (3). The lentogenic strains lack neurovirulence and do not form plaques in chicken embryo fibroblast (CEF) cell cultures (4,7). This can account for the scarcity of reported quantitative studies on lentogenic strains and for the fact that the few data available were derived almost exclusively from titrations in eggs (6). This paper describes plaque formation by lentogenic NDV strains in chicken embryo lung cell (CEL) cultures. This simple, rapid and reproducible procedure proved to be helpful for the titration and plaque purification of such strains. The lentogenic NDV strains LaSota, F, Ulster, and Queensland V4 were used. The origin and method of propagation of these strains were described earlier (5). CEL cultures were prepared from 14-day-old chicken embryos. Lung cells were obtained by digestion in 0.1% trypsin (Difco) and were seeded in TC Medium 199 (Difco) containing 5% calf serum (derived from colostrum-deprived calves). Cell monolayers were grown in petri dishes (Anumbra) 5 cm in diameter (3.5 x 106 cells per dish) and were used for titration after 20 to 24 h. The overlay medium contained 0.85% Special Agar-Noble (Difco) purified with ethylenediaminetetraacetic acid at pH 7.0, 5% calf serum, 1% of a 5% sodium bicarbonate solution, and the usual concentrations of penicillin and streptomycin in TC Medium 199. Plaque formation in CEL culture by the lentogenic LaSota, F, Ulster, and Queensland V4 strains is shown in Fig. 1. The plaques formed by lentogenic strains were 0.5 to 2 mm in diameter. A total of 14 lentogenic strains were examined, all of which formed plaques in CEL culture within 48 to 72 h. Embryonic chicken lung is a rich cell source, 45 to 55 x 106 cells being obtainable from each pair of lungs, and the cell monolayer required for plaque assay is more easily reproducible than with kidney cells. Barahona and Hanson (2) titrated lentogenic NDV strains in CEF culture in the presence of Mg2+ and diethylaminoethyl dextran. In our assay, five to ten times lower titers were regularly obtained with this method compared with titrations in CEL. Shingh et al. (8) lentogenic F strain and obtained plaques on the 4th to 5th day of incubation. It was found, however, that plaques formed in CEF cultures under agarose (Calbiochem) were turbid and not readable until 5 to 7 days after infection. Also, plaque counts were lower than in CEL culture, and certain strains had titers 2 to 3 logarithmic orders below the levels reached in CEL (Table 1). Although the lentogenic NDV strains are capable of forming plaques in embryonic kidney cell culture (1), these cells, unlike CEL, may be prone to degeneration. When samples of strains LaSota and Ulster were titrated in 9-day-old chicken embryos, values of 6.3 x 109 (101-1) and 2.5 x 109 (109.4) mean egg infective dose/ml were calculated, respectively; when the same dilutions of these strains were assayed in CEL, titers of 1.6 x 101 and 1.4 x 109 plaque-forming units/ml were obtained. Thus, plaque assay in CEL proved to be only 2 to 4 times less sensitive than in the egg system.

v3-fos

2020-12-10T09:04:22.683Z

1974-09-01T00:00:00.000Z

237230723

s2

Temperature-Induced Changes in the Sporicidal Activity and Chemical Properties of Glutaraldehyde Freshly prepared 2% acid and alkaline glutaraldehyde solutions were stored at 4, 20, and 37 C. At intervals, samples were removed and changes in pH, ultraviolet spectrum, and sporicidal activity (against Bacillus pumilus spores) were recorded. Alkaline solutions stored at 4 C showed little changes in these properties, whereas such solutions stored at 37 C became turbid and showed a decrease in pH, marked changes in ultraviolet spectrum, and an almost complete loss of sporicidal activity. Intermediate results were obtained with alkaline solutions stored at 20 C. In contrast, acid 2% glutaraldehyde solutions (initial pH 3.5) showed comparatively few changes in their properties. Treatment of spores with freshly prepared glutaraldehyde solutions (0.5%) at temperature above 40 C reduced the effect of pH on sporicidal activity. Glutaraldehyde is an important microbiocidal agent which is lethal at alkaline pH to bacteria, bacterial spores, fungi, and viruses (1, 4, 5-7, 9-12; S. Thomas and A. D. Russell, J. Appl. Bacteriol., in press), although specific tests conducted question tuberculocidal activity (9,10). Solutions of the dialdehyde are stable at acid pH, but when "activated" for use at alkaline pH their shelf life is short (1,12). We have been interested in the effects of temperature on the chemistry, physical properties, and sporicidal activity of glutaraldehyde, and results of these findings are presented in this paper. MATERIALS AND METHODS Glutaraldehyde solutions. Cidex, a 2% solution of glutaraldehyde, was kindly provided by Arbrook, Inc., Arlington, Tex. It was stored at 4 C. When required, 2 x 300-ml volumes were removed, one 300-ml portion being made alkaline by the addition of 0.3% (wt/vol) sodium bicarbonate. The 300-ml volumes of acid and alkaline solutions were subsequently stored at 4, 20, and 37 C, and at intervals samples were removed and monitored for pH, spectrophotometric analysis, and sporicidal activity against Bacillus pumilus spores, as described below. Spore preparation. Nutrient agar (Oxoid, Ltd., London, England) in Roux flasks was seeded with B. pumilus ATCC 27142 (E.601) and incubated for 5 days at 37 C. The growth was washed off the surface with sterile glass-distilled water and centrifuged, and the pellet was washed three times with sterile water and finally resuspended in sterile water to give 1010 to 1011 spores/ml. No preheating of the spore suspensions was made prior to sporicidal tests, since this may result in increased spore sensitivity to antibacterial agents (10). Sporicidal tests. A suitably diluted spore suspension (1 ml) was added to 2% alkaline glutaraldehyde (previously stored for the desired period at 4, 20, or 37 C) to give an initial spore level of approximately 10'0/ml. Samples were removed at intervals and, after dilution of glutaraldehyde to a sub-inhibitory level, viable survivors were determined by means of a plating method. The same procedure was adopted with the 2% acid glutaraldehyde solutions (previously stored for the desired period at 4, 20, or 37 C). Additional sporicidal tests were also made with these solutions, which were made alkaline by addition of 0.3% sodium bicarbonate immediately prior to inoculation with spores. pH changes. Changes in pH during storage at 4, 20, and 37 C of the 2% acid and alkaline glutaraldehyde solutions were recorded with the Cambridge pH meter. Spectral changes. Changes in the ultraviolet (UV) spectrum of the 2% acid and alkaline glutaraldehyde solutions during storage were recorded in the Unichem SP 800 spectrophotometer, using 1-cm cells and distilled water as the blank. The glutaraldehyde solution was diluted four times before examination. In some experiments, 0.5% solutions of acid and alkaline glutaraldehyde were allowed to equilibrate at elevated temperatures (40 to 95 C), and the absorbance was measured at 235 and 280 nm in a Hilger and Watts spectrophotometer fitted with a water-jacketed cell carrier. Sporicidal activity of glutaraldehyde at elevated temperatures. Acid or alkaline glutaraldehyde solutions (0.5%) were held at the desired temperature until equilibrium had occurred. They were then inoculated with bacterial spores, and samples were removed at intervals for viable counts as previously described. RESULTS AND DISCUSSION pH changes. Changes in pH of 2% alkaline glutaraldehyde solutions during storage at 4, 20, and 37 C are shown in Table 1. At 4 C, there was no change in pH over the 21-day period, a slight decrease with the solution stored at 20 C, and a more marked pH decrease with the solution stored at 37 C. In contrast, 2% acid solutions (initial pH 3.5) stored for 3 months at 4, 20, and 37 C had pH values of 3.6, 3.6, and 3.3, respectively, at the end of this period. Spectral changes. Ultraviolet spectrum changes of 0.5% alkaline glutaraldehyde solutions during storage at 4, 20, and 37 C are indicated in Fig. 1, which demonstrates that the solution stored at 4 C ( Acid 0.5% glutaraldehyde solutions stored at 4, 20, and 37 C showed a different response pattern (Fig. 2), in that negligible changes occurred at 280 nm even after a prolonged storage for several months at any one of these temperatures. There were, however, increases in the absorbance at 235 nm with increases in the storage period, especially at 37 C (Fig. 2c) and, to a lesser extent, at 20 C (Fig. 2b). In these cases, therefore, there was a significant increase in the 235 nm/280 nm ratio, which is believed to be an indication of increasing polymerization (8). Sporicidal activity. Two-percent alkaline glutaraldehyde, as a freshly prepared solution, is rapidly lethal to B. pumilus spores at 37 C (Fig. 3). In contrast, alkaline solutions stored at 37 C became cloudy and within a short storage period were no longer very actively sporicidal, whereas alkaline solutions stored at 4 C maintained their stability and anti-spore activity for considerably longer periods (Table 2). Intermediate results were obtained with alkaline solutions held at 20 C. Glutaraldehyde solutions are considerably less sporicidal and bactericidal at acid pH than at alkaline pH (1,5,8,11,12), and this point is again illustrated in Fig. 3, which shows the sporicidal activity of acid and alkaline 2% ately prior to spore inoculation were highly sporicidal, the viable spores per milliliter after 10 min of contact at 37 C being 6 x 103, 3 x 102, and 1 x 103 for solutions stored at 4, 20, and 37 C, respectively. One of the objections to using glutaraldehyde 200 250 300 350 as a disinfectant has been its comparatively WAVELENGTH NIM short shelf life after "activation." Storage of activated solutions at 4 C overcomes this criti-2.0 b 12 cism, and alkaline solutions are still highly active sporicidally even after storage for 18 months at this low temperature ( Table 2). /<1 \ Sporicidal activity and spectral changes at elevated temperatures. Figure 4 shows the effects of 0.5% acid or alkaline glutaraldehyde R 10 / / / \\ on bacterial spores at elevated temperatures. It is significant at temperatures above 40 C that the discrepancy in sporicidal activity between alkaline and acid glutaraldehyde is reduced, although it does not entirely disappear. There solutions at 37 C. Thomas and Russell (J. Appl. Bacteriol., in press) have previously shown that, le* at 20 C, 2% alkaline glutaraldehyde is considerably more lethal than a 2% acid solution, which exerts little sporicidal activity against these 5 10 15 spores over a 60-min period at this lower tem-TIME MIN perature. If, as seems possible, glutaraldehyde FIG. 3. Sporicidal activity of 2% alkaline (0) and acts as a protein cross-linking agent on the acid (-) solutions of glutaraldehyde against B. pumisurface layers of microbial cells (6,7,12), then it lus spores at 37 C. are two possibilities to account for this: (i) the higher temperatures cause some changes in the spores enabling greater penetration by the aldehyde, or (ii) changes in the dialdehyde molecule at alkaline pH occur on exposure to higher temperatures. Although the former may be a contributory factor, we have found a marked difference in the spectrophotometric properties of the acid and alkaline forms of glutaraldehyde with respect to temperature. Under acid conditions, the absorbance peak at 280 nm for glutaraldehyde increases markedly with increasing temperature (range tested, 40 to 95 C), whereas that at 235 nm remains relatively constant. Under alkaline conditions, however, the absorbance peaks at both 235 and 280 nm are greatly increased, the latter to the same extent as for the acid form. It has also been noted that the effect of temperature is reversible in the case of the 280-nm peak, but irreversible with the 235-nm peak when the alkaline glutaraldehyde is cooled to room temperature and reexamined. These results may appear to disagree with those of Rasmussen and Albrechtsen (8); however, the latter authors used long storage periods (up to 20 weeks). Thus, the differences in the properties of the two solutions could form a possible basis for explaining the differences in the sporicidal and bactericidal activity of glutaraldehyde at acid and alkaline pH. Overall comments. Two-percent alkaline glutaraldehyde is considerably more potent than acid glutaraldehyde as a sporicidal agent at 20 C (Thomas and Russell, J. Appl. Bacteriol., in press), but increasing temperature reduces the difference; above approximately 40 C there is little difference between 0.5% acid and alkaline glutaraldehyde solutions acting as sporicidal agents over a 60-min period. This conclusion supports that reached by Sierra and Boucher (11). There are, however, marked chemical changes which occur in alkaline glutaraldehyde solutions stored for periods of 3 weeks or more at 37 C, and these changes are reflected in a complete loss of sporicidal activity. The results also demonstrate the need for further research to be carried out on the chemical nature of the glutaraldehyde molecule, especially in relation to the nature and level of its sporicidal activity.

v3-fos

2018-04-03T06:18:20.225Z

1974-12-01T00:00:00.000Z

19708302

s2

Arthrobacter globiformis and Its Bacteriophage in Soil' Bacteriophages in soil for Arthrobacter globiformis were rarely detected unless the soil was nutritionally amended and incubated. In amended soil, phage were continuously produced for at least 48 h, and this did not require the addition of host cells. Rod and spheroid stage host cells added to the amended soil encountered indigenous bacteriophage, but added phage did not encounter sensitive indigenous host cells for some time, if at all. The indigenous phage in nonincubated soil seemed to be present in a masked state which was not merely a loose physical adsorption to soil materials but required growth conditions other than lysogeny for them to increase their titers. The possibility is discussed that the indigenous host cells in nonamended soil are present in a nonsensitive spheroid state, with the cells becoming sensitive to the phage in a rate-limiting fashion as nonsynchronous outgrowth occurs for a portion of the spheroid cells. Bacteriophages in soil for Arthrobacter globiformis were rarely detected unless the soil was nutritionally amended and incubated. In amended soil, phage were continuously produced for at least 48 h, and this did not require the addition of host cells. Rod and spheroid stage host cells added to the amended soil encountered indigenous bacteriophage, but added phage did not encounter sensitive indigenous host cells for some time, if at all. The indigenous phage in nonincubated soil seemed to be present in a masked state which was not merely a loose physical adsorption to soil materials but required growth conditions other than lysogeny for them to increase their titers. The possibility is discussed that the indigenous host cells in nonamended soil are present in a nonsensitive spheroid state, with the cells becoming sensitive to the phage in a rate-limiting fashion as nonsynchronous outgrowth occurs for a portion of the spheroid cells. Soil serves as a ready source for the isolation of virulent bacteriophage for many different bacteria (1). Little is known, however, about how these bacteriophage survive in soil and of the manner in which they locate and interact with their hosts in this habitat. Nor is it known how this interaction in soil is affected by pleomorphic growth cycles and dormancy states of the host bacteria. Answers to these obviously would be of interest from an ecological viewpoint, but, in addition, these answers might allow studies to be accomplished on the activities of specific bacteria in nature without isolating the bacteria. Thus, the specific bacteriophages for the bacterium are assumed to be present and operating in a defined manner in relation to their hosts, and these bacteriophages can easily be separated from the microbes and soil debris of the habitat and enumerated and evaluated by plaquing on host lawn plates of a specific bacterial species in the laboratory. The bacteriophage would thus serve as a natural internal indicator for the activities of a given bacterium in nature. Arthrobacter globiformis was chosen as the model bacterium for this study. It occurs naturally in soil (4) and, at least in the laboratory, it demonstrates a defined but not overly complex growth cycle (3,6,10,12,13). In addition, soil is known to harbor bacteriophages for this species (5,7,8,10). This bacterium and its bacteriophages were used, therefore, in the present ' This research was authorized for publication as paper no. 4746 in the journal series of the Pennsylvania Agricultural Experiment Station. study in an attempt to find answers to some of the more basic questions outlined above. MATERIALS AND METHODS Soil sample. A local Hagerstown silty clay loam (pH 6.0; plate count, 4.0 x 106/g at the time of use; moisture content, 25%; organic content, 3.5%; ref. 11) was used in this study. This sample consisted of the top 15 cm of soil collected directly beneath the surface vegetation, and it was stored in bulk for approximately 1 year in a sealed polyethylene bag. Media. Most experiments used BEG broth, which contained 0.3% beef extract and 0.5% glucose. The compositions of additional media are listed in Table 2. All media components other than sugars and inorganic salts were Difco products (Difco Laboratories, Detroit, Mich.). The synthetic medium included in Table 2 was based on Lochhead and Thexton (9) and contained glucose, 1.0 g; KHPO4, 1.0 g; KNOI, 0.3 g; MgSO4 7H2O, 0.2 g; CaCl2, 0.1 g; NaCl, 0.1 g; FeCl, .6HIO, 0.01 g; yeast extract, 1.0 g; and distilled water, 1,000 ml. The basal layer of phage-plaquing plates was BEG medium containing 1.5% agar; the overlay for these plates was the same medium but with 1.0% agar. A. globiformis cultures. The A. globiformis strains used in this study were American Type Culture Collection (ATCC) strains 8010 and 4336. Stock cultures were maintained on BEG medium containing 1.5% agar. Cultures of rodand spheroid-stage cells of this bacterium were grown by inoculating a loop of cells from a slant into 50 ml of BEG broth in a 300-ml baffle-bottom Erlenmeyer flask (Bellco Glass Inc., in distilled water for addition to soil, which was to be incubated at 60% of moisture-holding capacity (MHC), but were not washed or concentrated when used in broth studies. The lysogenic strain of 8010 occurred originally as a turbid plaque when a filtrate from a soil broth enrichment without added cells was plaqued with strain 8010 on nutrient agar. A culture recovered from this plaque was purified and then handled in a manner similar to that for the nonlysogenic strain. Soil incubation at 60% MHC. Portions (5 g) of soil in screw-cap tubes (18 [inner diameter] by 150 mm) were adjusted to 60% of the soil's MHC by adding distilled water plus a washed strain 8010 cell suspension (1.7 x 106 colony-forming units [CFUV5 g of soil) and/or a carbon or nitrogen solution nutritional amendment. The tubes were incubated with caps loose at 28 C for periods of from 0 to 4 days, and then the soil from each tube was added to 100 ml of BEG broth in a 300-ml baffle-bottom flask. This was shaken for 10 min and then centrifuged at low speed. The supernatant fluid, containing the phage, was passed through a membrane filter (0.3-Am pore size; Millipore Corp., Bedford, Mass.) and then further diluted in sterile BEG broth for phage plaquing. Broth-soil phage enrichments. Soil (4 g) was added to 100 ml of broth medium in a 300-ml baffle-bottom flask. In some experiments the flasks also received unwashed A. globiformis cells, either rod or spheroid stage, at a final concentration in the flask of 1.7 x 107 or 1.4 x 107 CFU/ml, respectively; the final flask titer of added FX-1 phage was 40 plaqueforming units (PFU)/ml and of added soil-mixed phage preparation was 100 PFU/ml. These flasks were shaken at 28 C, and, at various time intervals, 5-ml samples were removed and subjected to low-speed centrifugation. The supernatant fluid was then passed through a 0.3-,um membrane filter, and the filtrate was diluted in BEG broth for phage plaque assay. Two or more flasks per treatment were included so that an overall large withdrawal of fluid volume from any one flask during sampling would not occur to affect the results. Cell-phage interaction in absence of soil. To 80 ml of broth medium in a 500-ml Klett side-arm Erlenmeyer flask (Bellco Glass Inc., Vineland, N.J.) was added rodor spheroid-stage unwashed cells at a final concentration in the flask of 106 CFU/ml; the soil-mixed phage preparation, when added, was at a final titer of 50 PFU/ml. These flasks were shaken, and, at various time intervals, turbidity was measured as Klett units and 5 ml-samples were withdrawn and treated as above for phage plaque assay. For sonic treatment trials, phage suspension (1 ml), strain 8010 cell suspension (1 ml), soil (4 g), or mixtures of these were added to 100 ml of sterile BEG broth, and this was sonically treated for either 1 or 12 min in a Biosonic I oscillator (Bronwill Scientific, Inc., Rochester, N.Y.) operating at 112 W accoustic energy at the probe tip. The probe had been sterilized with alcohol. Phage sources and assay. Most phage samples for plaque assay were diluted in BEG broth. One milliliter of a dilution to be plaqued and 0.15 ml of strain 8010 broth culture (rod stage) were added to 2.5 ml of BEG 1% agar medium, and this was applied as an overlay on a BEG 1.5% agar basal layer. The experiments reported in Table 2, however, used nutrient broth and agar, respectively, for dilution and plaquing. The plates were incubated 48 h at 28 C, and then the plaques were counted. The plates were again observed at 4 days of incubation so as to note the occurrence of possible plaques not present at 2 days and whether any plaques had markedly increased in size. Periodic electron microscopy checks of phosphotungstic acid-negative stains were made of plaques resulting from soil enrichments to be sure that bacteriophage actually were causing the plaques. Virulent phage strain FX-1 was recovered from a soil enrichment (without added host cells) that had been plaqued on strain 8010 on nutrient agar. It was purified on this medium, and phage preparations were prepared by suspending the surface agar from confluent lysis plates in nutrient broth. The residual cells and agar were removed by low-speed centrifugation, and the phage suspension was passed through a sterile 0.3-aum membrane filter and refrigerated. The soil-mixed phage preparation was made as follows. Soil (12 g) was added to 300 ml of BEG broth in a 1,000-ml Erlenmayer flask. This was shaken for 24 h at 28 C and then clarified by low-speed centrifugation. The supernatant fluid was sequentially passed through sterile 0.8-and 0.3-am membrane filters to yield a mixed phage suspension containing 10' PFU/ml as plaqued on strain 8010. Phosphotungstic acid-negative stains of this preparation viewed by electron microscopy showed phage of several different morphologies, but no bacterial or other cells. RESULTS Soil present. We have not been successful in extracting bacteriophage for A. globiformis ATCC 8010 from our soil without first incubating the soil. Adjustment of the soil to 60% of its MHC with distilled water, with or without rod-stage host cell additions, and incubating at 28 C gave a few phage in one instance but none in the second instance (Table 1). Usually, however, no phage could be detected under these conditions. In contrast, incubation of soil amended with glucose or sucrose provided extensive phage production for this bacterium, and the response was greater when washed rod-stage host cells had been initially added to the soil. Several broth media were evaluated with the object of finding one that would more easily allow study of the phage enrichment process when host cells had not been added than was possible by incubation of the soil at 60% MHC. In addition, it was desired that the soil with its indigenous host cells and phage be, dispersed and agitated in a liquid medium, so that the effects of spatial discontinuities separating the phage from their hosts would be lessened. A Separate but similar experiment using 25 g of soil in polyethylene-covered glass tumbler. d Starch, glucose, and sucrose added at 50 mg/5 g of soil. e (NH4),SO4, NaNO., and urea added at 5 mg/5 g of soil. medium containing 0.3% beef extract and 0.5% glucose (i.e., BEG medium), inoculated and shaken with 4 g of soil (host cells not added), yielded 2.7 x 10' PFU/ml at 3 days of enrichment (Table 2). This medium, therefore, was used in most of the succeeding trials. BEG broths were inoculated with 4 g of soil, with and without additions of host cells or phage, and shaken 1 to 2 days at 28 C. The soil not receiving added phage or host cells continuously produced phage over the time periods tested ( Fig. 1 and 2). The added soil-mixed phage preparation, however, apparently encountered few if any sensitive indigenous host cells in the soil, and a delay in addition of the phage until 6 h of incubation had occurred did not change this picture. The pure laboratory strain of virulent phage (FX-1) did encounter some sensitive indigenous host cells in the soil (Fig. 2), but not until sometime between 14 and 24 h of incubation; this was shown to be a repeatable phenomenon. This in situ phage production by FX-1 was sensitive to sonic treatment. A 1-min sonic treatment at zero time of the broth containing the soil and FX-1 virtually eliminated this response. In contrast, the indigenous phage production occurring in the soil enrichment without added phage or host cells withstood a 12-min sonic treatment. Rod-stage cells of A. globiformis ATCC strains 8010 and 4336 responded alike in phage production when they were added to soil enrichments (Fig. 2). Note that the soil incubated with strain 4336 was plaqued on strain 8010. Both bacterial strains vigorously produced phage starting at about 8 h and extending to about 14 h of incubation. In contrast, the added spheroid cells of strain 8010 delayed phage production during the first 9 h (rate compared to the soil-only control; Fig. 1), but then continuously produced phage at least through 24 h of incubation. When the soil-mixed phage preparation and rod-stage host cells of 8010 were added simultaneously to the soil and the soil was incubated, the phage production curve closely resembled that obtained when only the bacteria were added without adding phage (see Fig. 1 and 2). Thus, in this case, the actual source of the phage inoculum (whether added or indigenous) producing the additional phage was not clear. being liberated in the soil as a result of growthrelated processes or simply were being physi-7.0 cally desorbed from a masked state on the soil debris, flasks containing distilled water or broth medium were incubated at 4 C, and filtrates prepared at intervals during incubation were 6.0 -enriched for 24 h at 28 C, as above, with a pure culture of strain 8010. The cold-temperature soil incubation comprised, sequentially, shaking for 13 To determine whether the phage were cells plus soil, genic strains occurred in heart infusion broth, and the growth of both strains was equivalent in this medium. The delay for growth initiation by the spheroid-stage cells was still present, however, in this medium. Figure 5 presents growth results and phage production resulting from lysogeny when lysogenic strain, spheroid-stage cells were used as inoculum; rod-stage cells were not tested for phage production. Addition of the h. These results for the rod-stage cells approximately correspond to those for rod-stage cells in the presence of soil ( Fig. 1 and 2), but the initiation of phage release for the spheroid-stage cells in Fig. 3 (absence of soil) seems to be delayed approximately 3 to 5 h as compared with the results in the presence of soil (Fig. 1). In the absence of added phage, both rodand spheroid-stage cells of the lysogenic strain produced almost no phage in this broth medium; 12 PFU/ml were detected at 18 h for the rod-stage cells, and 30 PFU/ml at 12 h for the spheroidstage cells. At other incubation times only 1 or 2 PFU/ml were detected. Both the lysogenic and nonlysogenic strains grew at a similar rate in this medium; Fig. 4 shows growth for rod-and spheroid-stage cell inocula of the nonlysogenic strain. As compared to BEG broth, considerably more growth of both the lysogenic and nonlyso- mixed-soil phage preparation did not materially change this picture for either growth or phage production. In contrast to these results, the spheroid-stage cells of the nonlysogenic strain (rod cells not tested) were virtually immune to added phage in the heart infusion broth. Growth as measured by turbidity was not altered, and the added phage were barely detectable or not detectable in samples taken during incubation. Both the rodand spheroid-stage cells of strain 8010 were quite resistant to sonic treatment; the CFU per milliliter were identical for cells not sonically treated and cells sonically treated for 12 min. The phage, however, displayed some sensitivity towards sonic treatment. Phage strain FX-1 showed 97% survival of PFU per milliliter with 1 min of sonic treatment and 43% with 12 min; the respective survival values for the soil-mixed phage preparation were 84 and 50%. Enrichment in shaken culture was not involved in these determinations, and soil was not present. The nonsonically treated, soil-mixed phage preparation was relatively resistant to the shaking involved in broth incubations; shaking in BEG broth in the absence of host cells or soil gave percent survivals of the phage at 5, 12, and 24 h, respectively, of 60, 35, and 31%. Rod-and spheroid-stage cells. Rodand spheroid-stage strain 8010 cells were used as inocula in these studies. The rod cells were from near the end of the logarithmic growth phase, and they presented a Klett value of approximately 256 units and a plate count of 1.7 x 109/ml. The respective values for the spheroid cells, which received two additional days of incubation, were similar, being 226 units and 1.4 x 109/ml. In addition to this age disparity, these cells differed in their morphology. The rod cells were short, gram-negative rods surrounded by what appears to be a slime layer. The spheroid cells were gram-positive coccoid rods slightly pointed at each end and surrounded by a thin, gram-negative casing. The delay in growth initiation observed when the latter cells were used as inoculum (see Fig. 4) seems to be due to the time required for pleomorphic outgrowth of these cells. The outgrowth at 12 h is shown in Fig. 6, which corresponds to a point on the growth curve (Fig. 4) when growth increase is not yet measurable as turbidity. DISCUSSION Bacteriophages for A. globiformis ATCC strain 8010 were rarely detected in our soil unless the soil was amended nutritionally and incubated. This was true for soil incubated with water at 60% of the soil's MHC and for soil shaken in water. Addition of washed host cells did not change this picture. In contrast, addition of glucose or sucrose to soil at 60% of MHC, with or without a simultaneous addition of host cells, stimulated a bacteriophage titer buildup to approximately 104 to 106 PFU/g of soil by the second day of incubation. Amendment of the soil with nitrogen-containing compounds, however, was less stimulatory to the production of phage. The sequence of events and the activities of the phage and host cells occurring during soil incubation were more easily studied by using shaken-aerated broth enrichments with soil, and, for approximately the first 24 h, these enrichments seemed to be representative of the 60% MHC soil incubations. Evaluation of several broth media showed that a medium comprised of 0.3% beef extract and 0.5% glucose (i.e., BEG broth) provided acceptable phage titers in the shaken soils as plaqued with A. globiformis ATCC strain 8010. When this medium was used for growing strain 8010 in the absence of soil, a reasonable growth rate and total amount of growth were obtained, although these were less than those obtained with a nutritionally richer medium such as heart infusion broth. Soil shaken in BEG broth (host cells or phage not added) started to produce phage which plaqued on strain 8010 at about 6 h of incubation, and this phage production continued for 48 h or longer. However, a decrease in the phage production rate usually occurred by about 24 h (Fig. 2). Host cells of strains 8010 and 4336 added to this soil enrichment encountered bacteriophage, as shown by phage titers greater than with the soil alone, starting at about 8 to 9 h of incubation. In contrast, phage added to the soil (host cells not added) did not seem to encounter sensitive host cells for some time, if at all. Laboratory strain FX-1 lytic phage interacted with the indigenous host cells in the soil starting some time after 14 h of incubation ( Fig. 2; rate compared with the soilonly control), but a soil-mixed phage preparation that had originally been recovered from this soil did not find sensitive host cells (Fig. 1). The reaction of the indigenous host cells in soil to the added FX-1 phage seemed to be a different phenomenon from that of the interaction of the indigenous phage and indigenous host cells already in the soil. Thus, sonic treatment of the mixture of soil and phage FX-1 before incubation destroyed the phage production response to this phage in the soil, but the phage production by the indigenous phage and were quite resistant to the sonic treatment levels used in this study, and that both the FX-1 phage strain and the soil-mixed phage preparation were relatively resistant to sonic treatment. The phage production in soil not receiving added host cells or phage did not seem to be merely a physical release of phage which had been masked by adsorption to soil materials, because phage titers did not build up in the soil when it was incubated in the cold. In addition, sonic treatment of the soil did not release additional phage over those in nonsonically treated soil. The strain 8010 cells used in this study were either gram-negative, short rods (rod-stage cells) taken near the end of their logarithmic growth or gram-positive spheroids (spheroidstage cells) from late in the maximal stationary phase of growth. On inoculation into fresh BEG medium, the latter cells delayed growth for approximately 6 h longer than did the rod cells. This extra time was required fdr-pleomorphic outgrowth of the spheroids (Fig. 6) before initiating a rapid growth phase. A lysogenic strain of 8010 was also used in these studies and, as concerns spheroid pleomorphic outgrowth and growth of the rod cells, it behaved in a manner similar to that of the nonlysogenic strain. The choice of bacterial cell numbers (either rod or spheroid stage, lysogenic or nonlysogenic) to be added to the soil or used as inoculum in pure culture studies was quite empirical because, although Arthrobacter species cells are thought to be quite numerous in soil (2), it was not known how many A. globiformis-like cells might be in soil that could respond to phage that plaque on strain 8010. Likewise, the numbers of phage PFU to be added to soil or pure bacterial cultures were open to question. Since the phage numbers recoverable from soil were nil or very low unless incubation with nutrients was employed, it was decided to use a low ratio of PFU to CFU in the pure culture experiments. The final ratio when soil was not present was approximately 1 PFU/2 x 10' CFU. A comparison of Fig. 3 and 4 for pure cultures in the absence of soil shows that, for both rodand spheroid-stage cells used as inoculum, phage production is initiated as the rod cells start to multiply and is rapid thereafter. The above-mentioned delay for spheroid pleomorphic outgrowth thus also applies as a delay for phage production. Figure 3, in addition, shows that the lysogenic strain in BEG broth with added phage responds with growth and phage production in a manner similar to that of the nonlysogenic strain. The delay for phage production by indigenous soil phage acting on spheroid-stage cells added to the soil, and the shorter delay for added rod-stage cells, are shown in Fig. 1. After these delays, however, phage production in both cases is rapid, with rates roughly equivalent to those resulting when 100 PFU of the soil-mixed prepa- VOL. 28, 1974 on March 23, 2020 by guest http://aem.asm.org/ Downloaded from ration per ml are reacted with 106 CFU of strain 8010 per ml (Fig. 3). These rates are considerably greater, however, than those for the interaction of indigenous phage with indigenous host cells ( Fig. 1 and 2). The latter rates could indicate that the sensitive host cell level naturally present in soil actually is quite low. From this viewpoint it is of interest that the 24-h indigenous phage production titers in Fig. 1 and 2 (host cells and phage not added) can be duplicated (experiment not reported) by shaking a mixture of 80 CFU of strain 8010 rod-stage cells per ml and 250 PFU of the soil-mixed phage preparation per ml for 24 h in BEG broth. A low available host cell number could also explain the inability of the added soil-mixed phage preparation to locate sensitive host cells, and of added strain FX-1 phage to find sensitive host cells until some time after 14 h of incubation. A low sensitive host cell level, however, does not necessarily mean that the total numbers of host cells are low. It is generally assumed that Arthrobacter species occur naturally in soil in the spheroid (coccoid) stage (4) and, although the present study has shown a defined time requirement for spheroid outgrowth, nothing is known about the frequency with which this outgrowth occurs in nature. In other words, the proportion of the spheroid population that does not respond with outgrowth to a given environmental stimulus is not known. A nonsynchronous spheroid outgrowth in nature, however, could provide a continuous but ratelimiting source of low numbers of cells sensitive to phage and, thus, might explain the present results. The survival of this bacterium in nature thus could well depend on there being a reservoir of spheroid cells which do not undergo outgrowth even though growth conditions may have improved. An alternate explanation for our results would be that the phage production in soil observed when neither host cells nor phage were added represents in total the results of lysogeny for the in situ cells or, alternatively, an initial production of phage through lysogeny with these phage then acting in a virulent manner on other sensitive host cells. These do not seem to apply in our study, and this conclusion is based on the opposing results obtained with the use of BEG and heart infusion broths, and on the fact that lysogeny as a means of inoculating sensitive host cells would impose too long a time delay before phage production could be initiated by the latter cells. Soil incubated in heart infusion broth (host cells and phage not added) produced only barely detectable levels of phage as contrasted with the phage yields produced in BEG broth (Table 2; Fig. 1 and 2). When soil was not present, the lysogenic strain of 8010 without addition of phage produced easily detectable levels of phage in heart infusion broth (Fig. 5), but barely detectable levels or no phage in BEG broth. Phage production in soil thus more closely resembles that associated with the nonlysogenic strain. Obviously, however, we used only one lysogenic strain in this study and, of course, do not know whether the soil harbors other lysogenic strains that would act in an entirely different manner under our experimental conditions. Based on the foregoing discussions taken as a whole, it would seem that in non-nutritionally amended soil the numbers of naturally occurring bacteriophage capable of plaquing on A. globiformis ATCC 8010 are low but not nil. Their numbers, however, cannot be precisely quantified, because they appear to be masked in some manner, other than through lysogeny or a loose physical adsorption to soil materials, so that they cannot be washed from the soil but, still, are available with the proper time delays for reaction with added rodor spheroid-stage host cells. The naturally occurring host cells in soil for these phage either are present in very low numbers or are present in a form insensitive to the phage. In the latter case, incubation with added nutrients would nonsynchronously change them into a sensitive state so that at any one time only a portion of the cells could interact with the phage.

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2020-12-10T09:04:12.839Z

1974-01-01T00:00:00.000Z

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Antibody to Viruses Affecting Cattle in Commercial Tissue Culture Grade Fetal Calf Serum Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum. Received.for publication 24 September 1973 Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum. Under normal conditions immunoglobulins are transferred from a cow to its calf solely via colostrum during the 1st day of a calf's life. There appears to be no evidence for intrauterine transfer as occurs in many other species (4). As the bovine fetus matures, it gradually develops immunocompetence so that it is able to respond to certain antigenic stimuli beginning at about the 118th day of gestation (16). Bovine viral diarrhea (BVD) (1, 10), bluetongue (3,12), and infectious bovine rhinotracheitis (IBR) (5,9) viruses have been reported to infect the bovine fetus by traversing the placental barrier. Antibody to these viruses has been found in infected fetuses (10,13,15). Recently, Hubbert et al. (8) tested the sera of apparently normal fetuses for antibody to the bovine strain of parainfluenza 3 (PI3-SF4), BVD, and IBR viruses. Antibody to these viruses is found in the majority of adult cattle; however, they only found antibody to BVD virus in 3 of 147 fetal sera and no antibody to either PI3-SF4 or IBR virus in about 100 fetal sera. Horner et al. (7) also tested the sera of individual fetuses. By using a variety of infectious agents as antigens, they found antibody only to BVD virus in a small percentage of the sera. Because of its availability, its growth promoting capacity, and its absence of immunoglobu-IPublication no. 1160, School of Veterinary Medicine, Auburn University, Auburn, Ala. 36830. lins, fetal calf serum (FCS) has been widely used in culturing cells of many species. However, Kniazeff et al. (11), noting that many investigators found that pooled FCS neutralized certain viruses, tested individual sera from bovine fetuses in their 4th to 6th month of gestation. They found that a few fetuses had antibody to BVD, but not to IBR or PI3-SF4 virus. The antibody detected in the fetus was attributed to either fetal synthesis or to transplacental transfer. Considering present information, transplacental transfer seems to be a remote possibility for explaining the presence of antibodies in the serum of bovine fetuses. Investigations of Boone et al. (2) showed that different lots of commercial FCS for use in tissue culture varied considerably in chemical composition. They suggested that high gamma globulin levels may be associated with adulteration with postcolostrol serum. In our work with bovine viruses, we have used the micro-serum neutralization (micro-SN) test in which virus and test serum are mixed and incubated together. Cells are seeded in this mixture so that this procedure requires a concentration of about 10% serum for suitable cell growth. Because of the requirement of serum for cell growth, antibody in the culture medium is a significant factor as compared with neutralization tests conducted on confluent cultures in which the concentration of serum in the medium is minimal. This report concerns the examination of commercial FCS for antiviral inhibitors to viruses affecting cattle to determine the suitability of different types and lots of FCS for use in the micro-SN system. MATERIALS AND METHODS Test serum. All tissue culture sera tested were from different serially numbered lots purchased from Grand Island Biological Company, Grand Island, N.Y., during the interval from the autumn of 1970 to the spring of 1973. The sera were heat inactivated nrid stored at -20 C until tested. The sera were identified by the manufacturer as (i) FCS, (ii) virus-screened FCS (VS-FCS), (iii) immunoprecipitin-tested FCS (IPT-FCS), or (iv) gamma globulin-free newborn calf serum (GG-free NCS). The latter two sera were subjected to a modified Cohn fractionation (6) procedure by the manufacturer to remove gamma globulins. Viruses. The following viruses were used in the micro-SN test: IBR, PI3-SF4, bovine adenovirus type 1, bluetongue strains 8 and OX 183 (BT 8 and BT OX 183), the New Jersey serotype of vesicular stomatitis (VS), and two bovine enterovirus strains identified as ED-1244 and 66-P-188 (supplied through the courtesy of J. Storz, Colorado State University, Fort Collins, Colo.). Micro-SN test. The micro-SN test was conducted as previously described (14) by using 50 to 100 mean tissue culture infective dose of virus, except for the following modifications. Medium for cells in microtiter consisted of Eagle minimal essential medium with modified Earle salts (Grand Island Biological Co.) and 10% IPT-FCS. The medium was buffered with 0.2% NaHCO3, 7.5 mM N-2-hydroxyethylpiperazine-N'-2'-ethane-sulfonic acid, and 5 mM each of TES and MOPS. Each serum was tested in triplicate by using serial twofold dilutions and starting with undiluted serum. A serum toxicity control was included for each dilution of serum. Complement enhancement test. Several sera which showed some inhibition of IBR virus, a herpesvirus, were tested with and without complement diluted 1:3 as described by Yoshino and Taniguchi (17), except that serum-virus-complement mixtures were assayed in microtiter by using six replicates per dilution. Complement has been found to enhance detection of antibody to IBR virus in certain bovine sera (C. R. Rossi and G. K, Kiesel, unpublished data). Cells consisted of a strain of bovine embryonic lung cells for assay of VS, IBR, BT 8, BT OX 183, and P13-SF4 viruses and the AU-BEK cell line, established in this laboratory (C. R. Rossi and G. K. Kiesel, In Vitro, in press), for assay of bovine adenovirus type 1 and bovine enteroviruses 66-P-188 and ED-1244. RESULTS AND DISCUSSION Results with the micro-SN test for detecting neutralizing substances in commercial sera against viruses which affect cattle are shown in Table 1. Low neutralizing titers against VS virus were found in most of the sera tested. The presence of neutralizing substances in IPT sera indicates the activity was not associated with the gamma globulin fraction of serum and suggests its non-antibody, nonspecific nature. Excluding VS virus, the five IPT-FCS tested did not inhibit replication of any other virus. At least two lots of IPT-FCS were toxic enough to prevent attachment and growth of bovine embryonic lung and AU-BEK cells when used at normal concentrations. This toxicity could be removed to allow satisfactory cell growth in the microtiter system by heating the serum at 56 C for 0.5 h. The GG-free NCS, one lot of FCS, and two lots of VS-FCS had inhibitory activity to several of the viruses, whereas most lots were completely free of any viral inhibitory activity. Explanations which can be offered for the presence of viral inhibitory activity in the commercial sera tested include the presence of (i) nonspecific inhibitors, (ii) fetal antibodies, (iii) natural antibodies, or (iv) antibodies due to contamination with post-colostral bovine serum or, in the case of GG-free NCS, ingestion of colostrum and incomplete removal of immunoglobulins. Serum lots 1, 3, 10, and 12 had inhibitory activity to viruses from several taxonomic groups, whereas most other lots of serum had no neutralizing activity against any virus, except for that against VS virus as previously mentioned. The distribution of neutralizing activity to several lots and types of sera tends to implicate antibody as the neutralizing substance detected against PI3-SF4, adenovirus type 1, and the two enteroviruses. Enhancement of neutralization to IBR virus by complement further indicates the specificity and antibody character of the neutralizing substances. The source of the antibody in these sera is probably contamination with serum from postcolostral bovine serum, in which these antibodies are common. The probability of fetal synthesis or natural antibody being distributed in the sera, as evidenced by the distribution of neutralizing activity in the sera, is remote. Because antibody was found to most of the test viruses in those lots of serum which contained antibody, it is likely that antibodies to many other viruses were also present in these same lots. Therefore, the micro-SN test would appear to be a useful technique for screening lots of commercial serum for antibody to bovine viruses and as an aid in identifying serum contaminated with post-colostrol bovine serum. The use of a few widespread bovine viruses should be appropriate in detecting such contamination. VOL. 27, 1974 adenoof para in-Vesicular bovine OX virus 66-P-ED-its hiora ACKNOWLEDGMENT This investigation was supported by the Alabama Agricultural Experiment Station, Auburn, Ala.

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2020-12-10T09:04:12.363Z

1974-01-01T00:00:00.000Z

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Effect of Amendments on the Microbial Utilization of Oil Applied to Soil Replicate field plots comprising a control, plus oil, plus oil and bacteria, plus oil and fertilizer (urea-phosphate; 27:27:0), and plus oil, bacteria, and fertilizer were monitored over a 308-day period for changes in bacterial and mold numbers. Changes in the chemical composition of the oil applied to the plots was followed by using chromatographic techniques. Application of fertilizer resulted in a stimulation of bacterial numbers and in the rate of utilization of n-alkane components of the saturate fraction. The application of oil-utilizing bacteria, however, resulted in only a slightly accelerated rate of utilization of n-alkane components of chain lengths C20 to C25. The isoprenoids, phytane and pristane, were still present in gas-liquid chromatography profiles after digestion of the n-alkane components of the saturate fraction. Those plots which received fertilizer showed an accelerated rate of recovery of native vegetation. Oil spills, whether on water or soil, do disappear (6), but very little is known about what can be done to accelerate this process. Recent work by Reisfeld et al. (5) and Atlas and Bartha (1) indicated that the disappearance of oil from sea water could be accelerated by the addition of deficient nutrients such as nitrogen or phosphorus, or both. Suggestions have also been made (4,7) for microbial seeding of spills since bacteria and fungi are the only biological species which have the metabolic capability of utilizing petroleum carbon for cell synthesis. There is, however, very little information in the literature evaluating the effect of such treatments on the acceleration of the utilization of oil spilled under natural conditions. Crude oil is essentially a mixture of carbon and hydrogen, and thus spills will result in an imbalance in the carbon-nitrogen ratio at the spill site. For bacteria to grow efficiently, they require about 10 parts carbon to 1 part nitrogen. If the ratio is greater, e.g. 100:1 or 1,000:1, growth of the bacteria and utilization of carbon source(s) will be retarded. In addition to there being a nitrogen deficiency in oil-soaked soil, other nutrients such as phosphorus may become growth-rate limiting. Therefore, in the experiments described in this paper, urea-phosphate, a fertilizer, was added to oil spilled on soil, thus correcting both deficiencies in one application. A survey of soils (unpublished observations) from the northwest area of Canada for the presence of oil-utilizing microorganisms indi-cated that not all soils have an indigenous population capable of utilizing oil. Thus, oil spills were also inoculated with oil-utilizing bacteria with and without a concurrent application of the urea-phosphate amendment. The experimental site chosen was in the Swan Hills area of north central Alberta, which represents a major oil-producing center in this province. The soils are of low fertility, are in a frozen state for approximately 5 to 6 months of the year, and are representative of soil and climatic conditions existing in the production and pipeline transport areas of this province and western Canada. MATERIALS AND METHODS Field sites. The plots in the Swan Hills area were placed on an overgrown, unused airstrip. Four replicate plots of each treatment, i.e., control, control plus oil, plus oil and bacteria, plus oil and fertilizer, plus oil, bacteria, and fertilizer, were placed in a random manner. The composition of the oil used in the spill is presented in Table 1. This crude petroleum was obtained from the Shell Oil Co. and is representative of producing wells in this area. The oil was applied in mid-July 1972 by sprinkling from perforated cans at a rate of 60 liters of crude oil per 9 square meters of soil, and it completely covered the surface of the plots with a thin layer of oil. Fertilizer, urea-phosphate (nitrogen-P20,-potassium, 27:27:0) was applied simultaneously to eight plots at a rate of 60 g of nitrogen per m2 (equivalent to 600 kg of nitrogen per hectare). A mixed culture of bacteria capable of utilizing an oil of similar quality (Norman Wells crude oil; chemical composition in Table 1) was also at this time applied b Used for growing cells which were applied to plots. c NSO, nitrogen-sulfur-oxygen-containing organic compounds. to eight plots, four of which had received a fertilizer treatment. The cells were grown on a rotary shaker (300 rpm, 1-inch [about 2.5 cm] eccentricity) at 25 C in 2-liter Erlenmeyer flasks containing 1 liter of basal salts medium (2) and 1 ml of Norman Wells crude oil as sole carbon source. The cells were recovered by centrifugation after 96 h of growth, washed, and resuspended in tap water at 4 C. This suspension was diluted to a concentration such that application of 6 liters of suspension per plot yielded an application rate of 101 bacterial cells per cm2. The mesophilic bacterial population used for bacterial seeding was composed of the following genera: Flavobacterium and Cytophaga sp. (41%), Pseudomonas sp. (34%), Xanthomonas sp. (10%), Alcaligenes sp. (9%) and Arthrobacter (5%). Soil samples (total weight approximately 500 g) were taken periodically and analyzed for total bacterial and fungal counts. When such samples were obtained from plots which received an oil treatment, the oil was extracted and its chemical composition was determined by chromatographic techniques (2). Thus, this experimental protocol allowed the statistical analysis of the effect of treatments on the microbial population and on the utilization of the applied oil. Microbiological methods. Changes in microbial numbers were monitored by using a spread plate count technique. Plate count agar (Difco) was used for the enumeration of bacteria, and malt extract agar (Difco) adjusted to pH 4.5 was used for molds. Quintuplicate plates of each dilution were incubated at 21 C for 6 days before counting. The bacteria which comprised the mixed population were classified to the generic level on the basis of the following tests: Gram reaction, presence and position of flagella (determined by electron microscopy); oxidation and/or fermentation of sugars with and without acid and/or gas production; catalase and oxidase activity. Isolates which were classified as Pseudomonas were streaked on Pseudomonas F and P agar (Difco) and observed for fluorescein or pyocyanin production, respectively. Chemical methods. The chromatographic techniques used for the analysis of crude oil was as described in our previous paper (2). These techniques resolve crude oil into asphaltene, saturate, aromatic, and the polar nitrogen-sulfur-oxygen-containing organic fractions. The saturate fraction was further resolved by using gas chromatography as reported previously (2). Recovery of oil from soil. Approximately 100 g of a soil sample was extracted four times with 100-ml portions of n-pentane, and the extracts were combined and evaporated to dryness in a fume hood at room temperature. The dry residue was redissolved in 4-to 25-ml portions of benzene, and, after pooling the benzene fraction, non-hydrocarbon material present was allowed to settle out. A portion of the benzenesoluble fraction was evaporated to dryness, weighted, resuspended in n-pentane, and analyzed by liquid and gas chromatographic procedures (2). Statistical analysis. Before statistical analysis, all data were subjected to the Nalimov test (3), which rejected data outside of the confidence limit of 95%. The means and t tests were made between sets, each possessing a minimum of four observations. All statistical analyses were performed on a minimum of four observations. All statistical analyses were performed by an IBM 360 computer. RESULTS The gas-liquid chromatography (GLC) profile of the n-saturate fraction of the oil before and immediately after application to soil is presented in Fig. 1. The mean pH values for these plots 12, 66, and 308 days after treatment ranged from pH 4.8 to 5.9. Statistical analysis of these data indicated that there were no significant differences between plots as a result of amendment application. Changes in the bacterial count observed 12, 66, and 308 days after treatment are presented in Table 2. These data show that there was a statistically significant increase in bacterial numbers within 12 days of the application of oil when fertilizer had been applied to the plot as well. The stimulation of bacterial numbers decreased within 66 days of treatment, and by 308 days the values for those plots that had received fertilizer, with or without bacteria, were similar. Statistical analysis of these results ( Table 2) indicated a consistent significant difference at the 95% confidence level and occasionally at the 99% confidence level when the effects of the fertilizer amendments on bacterial counts were compared with those values obtained from the control oil plots. Changes in the mold counts are presented in Table 3, and statistical analyses indicated that the application of amendments was without effect on this parameter at the 95% confidence level. The chemical composition of the oil recovered from the plots 12 days after treatment is presented in Table 4. Statistical analysis of these data showed that the application of amendments was without effect on the chemical composition of the oil recovered from the plots. The data in Tables 5 and 6 suggest that the oil recovered 66 and 308 days after treatment from plots which received the fertilizer amendment have a reduced n-saturate content. Statistical _ analysis of this reduction in n-saturate content of the recovered oil indicated a significant reduction (at both the 95 and 99% confidence levels) 66 and 308 days after treatment in those plots which had received a fertilizer treatment. A comparison of the n-saturate profiles 12 days after the oil had been applied to the soil (Fig. 2) shows that the application of fertilizer resulted in a slightly accelerated rate of n-saturate utilization. The change in profile is reflected in a utilization of the middle chain-length Df the satu-(i.e., C18 to C29) n-saturate components. The and after data in Fig. 3 show that the fertilizer application resulted in a complete disappearance of the n-saturate components, with the exception of U1s plots the branch-chain isoprenoids, phytane and pris-10/g after a tane, 66 days after treatment. These data also 308 days suggest a stimulatory effect by the application -308 days of the bacteria at this time which is reflected in _ an increased utilization of the C20 to C25 41.7 chain-length n-saturate compounds. These re-60. 1 suits are confirmed in Fig. 4, where a compari- dition can be responsible, in part, for the persistence of oil applied to soil has been substantiated by the data presented in this paper. The application of nitrogen and phosphorus as urea-phosphate resulted not only in a rapid increase in the numbers of bacteria present but also in an accelerated rate of disappearance of the n-saturate fraction of the crude oil applied to the soil. However, the efficacy of the application of oil-utilizing bacteria is still unanswered at this time since the effect reported, i.e., the slightly accelerated utilization of n-saturates of chain lengths C20 to C25, was not observed on all replicates. The low degree of change observed between the 66-and 308-day samples can be accounted for by the fact that for most of this period the area was in a frozen state. The persistence of the isoprenoids, phytane and pristane, in the GLC profile of those plots where n-saturation utilization occurred (i.e., where fertilizer was applied) rate fraction when fertilizer had been added and an accelerated rate of utilization of the C20 to C25 group of n-alkanes when bacteria had been applied to the plots. DISCUSSION The hypothesis that a nutrient-deficient con- suggests that psychrophilic conditions prevail in the soils in this area. It has been noted (2) that these compounds are more recalcitrant to microbial attack under psychrophilic than under mesophilic conditions. The slight difference in the chemical composition of the oil recovered from soil and that obtained from the barrel was probably a result of our extraction procedure. However, the procedure, at least for the major components of oil (i.e., saturates and aromatics) gave very highly reproducible results (Fig. 1). Initial studies were performed comparing n-pentane, benzene, methanol, and methylene chloride as extractives for recovering oil from soil. All of these solvents readily recovered the n-saturate fraction of oil applied to soil, but only n-pentane did this without removing residual soil material which subsequently interfered with our analytical procedures. For example, the material extracted from the soil by the other solvents was subsequently extracted into the benzene fraction and thus effected the further purification of the oil. The increase in the insoluble fractions when fertilizer had been applied to the plots (for example, the insoluble asphaltenes) suggests that transformation of oil components is taking place along with the assimilation of the n-satu-rate fraction. Changes in polarity of components result from the introduction of oxygen into the various components. Since our liquid chromatographic separation procedure is based on polarity, this change would shift these compounds into more polar fractions. At this time, however, it is not possible to account for the increase in total asphaltenes which were detected in the oil recovered from the plots 308 days after treatment. The lack of stimulation of the mold population as determined by our plate count technique is possibly an artifact resulting from our procedure, or more likely it reflects the inability of molds to successfully compete with bacteria under the nonrestrictive environmental conditions found in our plots. The slight increase in the rate of oil utilization by the application of bacteria to the spills could be a result of too low a level of application or to the inability of these bacteria to survive under the natural field conditions existing in the Swan Hills area. However, considering that nc'C FIG. 4. Gas chromatographic profiles of the saturate fraction of the Swan Hills oil 308 days after contact with the soil and various amendments. the bacterial content of these soils is 10-fold higher than what we applied, the failure to obtain a clear-cut effect is probably a result of too low a level of application. The partial utilization of n-saturates observed 308 days after application in those plots where oil alone was applied indicates that some component(s) of the indigenous flora present in the Swan Hills soils has the capability of utilizing crude oil. Visual observation of the rate of recovery of plant growth indicates an accelerated rate of recovery for those plots which received fertilizer. Thus, an increase in bacterial numbers can be correlated with an increased rate of disappearance of the n-saturate fraction component of crude oil, and this latter observation can be correlated with an increased rate of recovery of vegetation. All of these effects can be directly related to the application of fertilizer (ureaphosphate) to oil-soaked plots.

v3-fos

2020-12-10T09:04:20.693Z

1974-09-01T00:00:00.000Z

237229158

s2

Rice-Grown Rhizopus oligosporus Inoculum for Tempeh Fermentation A method of growing Rhizopus oligosporus on cooked rice as the inoculum for the fermentation of soybeans into tempeh was described and evaluated. Isolated R. oligosporus spores on glass beads survived best at low temperature and intermediate humidity. The activity of the rice-grown inoculum to ferment soybeans into tempeh did not decrease appreciably when stored desiccated for one year at 4 C or room temperature. Bacterial contaminants as high as 108 counts per g of cooked soybeans did not seem to affect the fermentation. A method of growing Rhizopus oligosporus on cooked rice as the inoculum for the fermentation of soybeans into tempeh was described and evaluated. Isolated R. oligosporus spores on glass beads survived best at low temperature and intermediate humidity. The activity of the rice-grown inoculum to ferment soybeans into tempeh did not decrease appreciably when stored desiccated for one year at 4 C or room temperature. Bacterial contaminants as high as 101 counts per g of cooked soybeans did not seem to affect the fermentation. Tempeh is a popular Indonesian food made from fermenting soybeans with the Rhizopus mold. Besides the academic interest in the fermentation process, the study of tempeh has been stimulated by the prospect of large-scale production that will be important in providing a low-cost protein diet. Methods of tempeh production in a traditional cottage scale vary in several details (1,7,10,11,13). Essentially, air-dried soybeans are soaked in water and the seed coats are removed. The cotyledons are steamed or boiled in water, drained and cooled, and inoculated with one of the several traditional mold inocula. The beans are then packed in small parcels and incubated at room temperature (25 C) for approximately 40 h. Fermentation is considered complete when the beans have been bound tightly by the mold mycelium into compact white cakes, which are customarily consumed within a day or two. Traditionally the inoculum is obtained in several ways. It may be the mold residue left over in the wrappings of the previous tempeh cake (1), or tempeh itself may be broken into pieces and mixed with the prepared soybeans (10). Pulverized dried tempeh can also be used (9). The surface of a tempeh, where most of the mold mycelium is found, may be sliced and sun-dried to be used as an inoculum. from growing the mold on rice and cassava have also been reported (3). Hesseltine (4) found Rhizopus oligosporus Saito to be the principal mold species responsible for the fermentation. S. D. Ko isolated molds from more than 80 tempeh samples collected throughout Java and Sumatra and found that this species was always present in tempeh of good quality (unpublished data). Pure cultures of this species have been used in a number of laboratory studies (5,6,8,14) as well as in a pilot plant study (12). In studies to improve traditional methods of tempeh production, our laboratory in Bandung, Indonesia, developed a simple but reliable method of inoculum preparation. In this method R. oligosporus was grown on cooked rice, which was then dried and used as the inoculum. The method was evaluated, the viability of isolated spores and spores in the inoculum was determined, and bacterial contamination during fermentation was examined. MATERIALS AND METHODS Rice. Locally bought sun-dried rice was used. Depending on the variety, the rice was cooked with 1 to 1.5 times its weight of water to obtain lumps that could be separated easily. Mold culture. Throughout the experiments R. oligosporus NRRL 5865 was used. It was originally isolated from a tempeh sample bought in a market in Bandung, Indonesia. Culture media. Ten grams of mungbean sprouts (Phaseolus radiatus) was extracted by boiling in water for 2 h, and the final volume of the extract was made to 100 ml. The medium, taoge sucrose agar, contained 6% sucrose and 2% agar in a 10% mungbean sprout extract. Taoge dextrose contained 1.5% dextrose in a 10% mungbean sprout extract. Preparation of the rice-grown mold inoculum. Sterile water (5 ml) was added to a pure culture of R. oligosporus grown on a taoge sucrose agar slant for 7 days at 37 C. The spores were scraped off the agar with an inoculating wire. One milliliter of the spore suspension was mixed with cooked rice obtained from every 100 g of sundried rice. The inoculated rice was spread to a loose layer approximately 1 cm thick in a covered aluminum tray (31 by 11 by 1.5 cm). The bottom and lid of the tray were perforated (1-mm-diameter holes, 15 mm apart) (Fig. 1). The tray was incubated at 37 C in a standard incubator without air circulation for 8 days to obtain a dry inoculum. The inoculum so obtained could also be used to inoculate a new batch of preparation. For this purpose, 1 g of the inoculum was mixed with cooked rice obtained from every 100 g of sun-dried rice. Storage of spores. Two spore preparations were stored in different combinations of temperature and humidity to test viability. These preparations were: (i) spores adhered on glass beads prepared by rolling 30 glass beads of 5-mm diameter (wetted aseptically with 50% sucrose solution) over a sporulating 3-dayold mold culture grown in a petri dish of taoge sucrose agar at 37 C; and (ii) the dried inoculum crushed in a mortar into pieces of about 5 mm. Thirty glass beads or 20 g of inoculum pieces were placed in an uncapped, 60-ml cylindrical glass jar. The jar was placed inside a capped, 240-ml cylindrical glass jar. To provide a specific relative humidity (RH), the bigger jar contained 40 ml of water (100% RH), 40 ml of 44% HS04 (approximately 50% RH), or 20 g of granular CaCl2 (near 0% RH). These materials were replaced every 4 weeks. The jars were stored in a 45-C incubator, in a shelf in the laboratory (25 C), or in a 4-C refrigerator. Determining spore viability. Germination of the spores was the criterion for spore viability. At 2-week intervals, one glass bead or one inoculum piece from each storage condition was shaken in a test tube containing 2 ml of taoge dextrose. The suspension from the inoculum piece was allowed to settle for 30 s, and the supernatant was decanted into another test tube containing 2 ml of taoge dextrose. The final suspensions were incubated at 37 C for 6.5 h. Germination percentage was calculated in triplicate from total spore count (approximately 500) and germinating spore count in 10 microscopic fields of the suspension placed in a hemacytometer. Calculated germination percentages fluctuated (within 5% germination) from one interval to the other. Best-fit curves were drawn through these fluctuations ( Fig. 2 and 3). RESULTS AND DISCUSSION Cooked rice consistency. The rice should be cooked to obtain easily separated small lumps so that the mold can grow on as much substrate surface as possible. A common mistake is to use too much water. This results in poor spore production, since the mold can only grow on the outer surface of the sticky rice mass. Process development. Before incubation, the inoculated rice contained 106 to 10" mold spores per g. During the first day of incubation, the spores germinated and the subsequent growth covered the rice lumps with a white, wooly mycelial layer. On the second day the color turned to light gray due to the formation of sporangia. During the following days more sporangia were formed and the color of the mass changed to dark gray. Concomitant with the darkening of the color, the initial substrate moisture content of 67% gradually decreased to 5% or less at the end of the 8-day incubation. Due to this dehydration process, the rice mass gradually shrank and became crusty (Fig. 1). The final product contained 108 to 109 mold spores per g. Application. For every kilogram of the original air-dried soybeans, 1 g of the inoculum was used. The inoculum was crushed into small pieces or pulverized, and mixed with the cooked soybeans. The methods of inoculum preparation and application were successfully testedin a pilot plant producing 75 kg of tempeh daily for more than 1 year. Spore germination percentage during storage. At the time the spores were prepared, the germination percentages were 72% for spores adhered on glass beads and 69% for spores in the inoculum pieces. These numbers decreased rapidly during the early storage period. Thereafter, in conditions favoring the survival of the spores, the germination percentage leveled off for some time, depending on the storage condition, and subsequently declined to very low points. Changes in germination percentages of the spores on glass beads are shown in Fig. 2. The curves can be associated into three distinct groups, each belonging to the same temperature condition. The lower the temperature the longer the spores survived. At 45 C, survival of the spores was favored by 0% RH. At 25 and 4 C, however, intermediate humidity (25% RH) was more beneficial than the two humidity extremes (100 and 0% RH). Thus, the longevity of isolated R. oligosporus spores are maintained best at low temperature, as is true for most fungal spores, and the spores can be classified among those whose survival is greatest at intermediate relative humidity (2). Changes in germination percentage of spores in the inoculum are shown in Fig. 3. Combinations of high temperature and/or high humidity (45 C-100% RH, 45 C-50% RH, 25 C-100% RH, 4 C-100% RH) rapidly decreased the germination percentages during the first few weeks. At high humidity the inoculum absorbed moisture and clumped together. High temperature black- at 45 C-O% RH and 25 C-50% RH started to decline, whereas the germination of those stored at 25 C-0% RH, 4 C-50% RH, and 4 C-0% RH stayed almost at the same level of 30 to 40% germination to the end of the 60-week experiment. Keeping quality of the inoculum. At the end of the above experiment, those inocula stored in the last three combinations were used to make tempeh. No discernible differences in the fermentation time or the quality of the tempeh were observed as compared with tempeh made with freshly prepared inoculum. This was also true for inocula stored for 1 year in a stoppered bottle and in sealed plastic bags. The results indicate that the best storage conditions for the preservation of the inoculum are low temperature (4 C) and low relative humidity (near 0%). When refrigeration is not available, the inoculum can be stored in sealed dry containers at room temperature. Bacterial contaminants. The rice and soybeans were cooked without pressure, and after cooking both showed bacterial counts of 10o to 104/g. Cooked rice inoculated with a previous batch of inoculum showed an increasing count during the first 2 days of incubation, when the moisture was still above 35%. Thereafter it decreased to 104 to 105/g in the final dried inoculum. Notwithstanding the bacterial population, tempeh fermentation was not disturbed if normal hygienic procedures were followed. In separate experiments, cooked soybeans inoculated with pure cultures of R. oligosporus were deliberately contaminated with one of the following arbitrarily chosen bacteria: Bacillus mycoides, Escherichia coli, Pseudomonas cocovenenans, Pseudomonas pyoceanea, and Proteus sp. In spite of counts up to 2 x 108 of these bacteria per g, tempeh of regular quality was obtained. An antibacterial agent has been found in tempeh extracts (15), and this may play a role during fermentation. Presumably, before the bacteria can proliferate, fermentation is complete and the tempeh is deep-fried or cooked in soups. No attempts have yet been made to identify the bacterial species in cooked rice or cooked soybeans, nor have the development of these bacteria and their effects on tempeh fermentation been studied.

v3-fos

2020-12-10T09:04:23.007Z

1974-07-01T00:00:00.000Z

237231708

s2

Physiology of Sporeforming Bacteria Associated with Insects: Minimal Nutritional Requirements for Growth, Sporulation, and Parasporal Crystal Formation of Bacillus thuringiensis A defined medium is described in which 18 strains of Bacillus thuringiensis representing the 12 established serotypes grow, sporulate, and produce a parasporal crystal. This minimal medium contains glucose and salts supplemented with either aspartate, glutamate, or citrate. These organic acids are required and cannot be replaced by vitamin mixtures or succinate even though succinate is taken up at a rate similar to that of aspartate, glutamate, and citrate. Bacillus thuringiensis, a bacterium found in natural association with lepidopterous insects, differs from most other sporeformers by synthesizing a discrete parasporal protein crystal in addition to the endospore. The importance of this crystal to the physiology of B. thuringiensis is obvious when one considers that it often constitutes 30% of the sporangium dry weight (16). Delafield et al. (7) postulated that the parasporal crystal arises from an unregulated superproduction of spore coat protein. The parasporal crystal is toxic to insects and, consequently, B. thuringiensis has become an economically important microbial insecticide (16). Use of the organism as such has considerably outpaced our understanding of the cause of parasporal crystal formation, the chemical nature of crystal structure, the mechanism of toxicity, and the control of crystal formation vis-A-vis sporulation. Particularly, there is a lack of information on the nutrition and physiology of B. thuringiensis relating to growth, sporulation, and crystal formation. In this communication and the accompanying report (15), we present results of comparative nutritional studies with 18 strains of B. thuringiensis. These strains include the 12 recognized serotypes and esterase types (5,6,11,12). Serotype denotes the presence of a specific flagellar H antigen, whereas the esterase type refers to an electrophoretic variant. To our knowledge, there are no reports comparing nu- ' MnSO4.H20, 0.05 g; CaCl2, 0.08 g; ZnSO4.7H20, 0.005 g; CuSO4.5H20, 0.005 g; FeSO4-7H20, 0.0005 g; K2HPO4, 0.5 g; (NHj),SO4, 2.0 g; and 1.0 g of glucose per liter of distilled water (pH adjusted to 7.4). BM is essentially the G medium devised by Nakata and Halvorson (14) for B. cereus, except that yeast extract is omitted. All chemicals used were reagent grade. The glucose and phosphate were autoclaved sepa-rately and added aseptically before inoculation; all vitamins were filter-sterilized. Cultures (10 ml) were grown at 28 C in 25-ml Erlenmeyer flasks and aerated by rotary agitation at 250 rpm. Inocula were prepared as previously described (2) by using BM plus glucose and glutamate. Radioisotope uptake studies. B. thuringiensis var. entomocidus (B-4046) was grown on BM plus 0.2% glutamate. Vegetative cells were harvested and washed twice in BM plus 0.02% chloramphenicol. These cells were suspended in the same medium at a final concentration of 1.0 mg (dry weight)/ml and equilibrated for 2 min at 28 C. Radioactive substrate (1 IACi) and carrier substrate were added to bring the final volume to 5 ml. Samples (1 ml) were removed at 2, 5, 10, and 15 min, filtered on pre-wetted 25-mm membrane filters (0.45 IA) (Millipore Corp.), and washed twice with 5-ml portions of BM plus 0.02% chloramphenicol. The filters were transferred to vials containing 10 ml of scintillation fluid (20) and were counted in a liquid scintillation spectrometer. Zero time radioactivity, determined in the absence of cells for each substrate, was subtracted from the resulting values. RESULTS It should be emphasized at the outset that B. thuringiensis does not grow in minimal glucosesalts media. Unsupplemented BM did not support growth of any of the 18 strains listed in Table 1. However, all strains grew and sporulated when BM was supplemented with either citrate, aspartate, or glutamate ( Table 2). Crystal formation always accompanied sporulation, and these crystals were toxic to lepidopterous insects (H. T. Dulmage, personal communication). Each strain grew well when BM was supplemented with either citrate, aspartate, or glutamate at a concentration of 0.2%. This substrate concentration is greater than that normally required to fulfill other amino acid auxotrophic requirements. Guirard and Snell (9) concluded that non-bacilli require amino acids at a concentration of about 0.001%; however, the bacilli may require abnormally large amounts of glutamate in chemically defined media. The defined medium devised by Nakata (13) for B. cereus contained 0.184% (12.5 mM) glutamate, and Buono et al. (3) reported that 1.0% (68 mM) glutamate is required for sporulation of B. cereus. If an intact tricarboxylic acid cycle is present, the carbons of exogenously supplied aspartate and glutamate should equilibrate with those of citrate and the other intermediates of the tricarboxylic acid cycle. Table 2 reveals that when BM was supplemented with either citrate, mal- obvious explanation for the minimal aspartate and glutamate concentrations that support growth (Table 3); these concentrations are well above those necessary for strictly metabolic purposes if the substrates had unrestricted access to the cell interior. Also, because Ghei and Kay (8) reported that the dicarboxylic acid transport system in B. subtilis is less efficient for succinate and fumarate, we suspected that the inability of succinate to promote growth ( Table 2) was also due, in part, to a transport problem. Accordingly, we conducted uptake studies (Fig. 1A-D) using radioactive aspartate, citrate, glutamate, and succinate. All four substrates were taken up by B. thuringiensis, but only the citrate and succinate pools showed saturation during the time periods employed. The pool sizes observed are in excellent agreement with those reported by Ghei and Kay (8) for B. subtilis. Figure 1A demonstrates that succinate does indeed have access to the cell interior, and we had to search for another reason for its inability to promote growth. We considered the possibility that succinate might be toxic to B. thuringiensis when present at concentrations as high as 0.2%. Succinate could have an adverse regulatory effect only evident when present in abnormally large amounts (17 mM). Unique regulatory phenomena have been frequently observed in B. thuringiensis (4,16). This possibility was eliminated by the following observations: (i) BM plus only 0.02% succinate did not support growth; (ii) when B. thuringiensis var. entomocidus was grown at a wide variety of succinate-glutamate ratios, all glutamate concentrations that allowed growth in the absence of succinate also allowed growth in the presence of up to a ninefold excess of succinate; (iii) similarly, succinate did not prevent the citratestimulated growth. Glucose uptake can be selectively inhibited under certain nutritional conditions. Romano and Kornberg (17) found that growth of Aspergillus nidulans on an acetate-containing medium inhibited glucose uptake, whereas sucrose uptake and catabolism remained unaffected. With this in mind, we modified BM by eliminating the glucose and replacing it with either sucrose, lactose, or mannitol. These modified BM also did not allow growth. Further, when B. thuringiensis was grown on BM plus aspartate or glutamate, glucose was taken up and catabolized via the Embden-Meyerhof-Parnas pathway (15), the same as it would be in yeast extract-grown cells. The presence of glucose is not, however, essential in most cases. Table 2 shows that most strains were capable of growing when glutamate or citrate were added to a glucose-free BM; the absence of glucose generally resulted in defective sporulation. DISCUSSION We have developed an appropriate chemically defined medium suitable for various biochemical, genetic, and physiological studies of B. thuringiensis. All of the strains not requiring vitamins grow and sporulate in our BM with the addition of 0.2% citrate, aspartate, or glutamate. This medium is a significant improvement over the two defined media previously reported (4,18). That of Singer et al. (18) is basically a glucose-salts medium, whereas that of Conner and Hansen (4) contains citrate as the sole carbon and energy source. Neither medium is adequate; each allows only minimal growth in just a few strains. In addition, sporulation is very poor in those strains that do exhibit growth. The contention that B. thuringiensis var.. berliner grows in glucose-salts media (16,18,19) is misleading because the "salts" used in those media included 0.0016% ferric ammonium citrate, 0.005% zinc acetate, and 0.01% ethylenediaminetetraacetic acid. The citrate and ethylenediaminetetraacetic acid may have been responsible for the slight growth observed by Singer et al. (18). The inability of succinate to promote growth when added to BM remains unclear. The succinate is not toxic, and uptake studies demonstrate that it does have access to the cell interior. We are currently investigating the possibility that succinate cannot be further metabolized in BM. This phenomenon is suggested by the absence of a continued increase in succinate uptake with time (Fig. 1A), unlike that occurring with aspartate (Fig. 1C). In addition, radiorespirometry using radioactive succinate (K. W. Nickerson and L. A. Bulla, unpublished data) shows that cells grown in BM plus 0.2% glutamate do not oxidize succinate to carbon dioxide, whereas under identical conditions yeast extract-grown cells do (1). Citrate is able to promote growth in the lowest concentrations of those substrates tested (Table 3) and yet is taken up least by the cells (Fig. 1B). This observation suggests that citrate may be the essential component. Aspartate and glutamate work just as well because they can be converted to citrate, whereas this conversion is unavailable to succinate. Attention must be paid to the pitfalls encountered when conducting nutritional studies on sporeformers. A medium may be sufficient to support vegetative growth and yet not be sufficient for germination and outgrowth of spores. BM plus 0.2% glutamate gave excellent growth in every case, and yet the timing of spore germination in this medium was erratic. The vitamin-free Casamino Acids starter culture represented a compromise in that the medium had to be rich enough to promote prompt spore germination and yet defined enough to allow rapid detection of vitamin and other nutritional requirements on subsequent transfer. It is important to examine each inoculum microscopically to determine whether the cells are still vegetative. An inoculum committed to sporulation would test a medium's ability to support sporulation, germination, and outgrowth, in addition to vegetative growth. It is much harder to define nutritional requirements for sporulation because a medium is no longer chemically defined after vegetative growth has been completed. A distinction between the ability to sporulate and the ability to form stable spores requires frequent microscope observation. Dormant spores are stable and may be observed any time after growth has been completed. However, spores unable to maintain dormancy may lyse following premature germination and outgrowth. Absence of or reduction in the number of cellular forms can mean either that the cells lysed without ever sporulating or that defective spores were formed. The media described in this report produce stable dormant spores.

v3-fos

2017-06-19T18:32:03.201Z

1974-04-15T00:00:00.000Z

5396338

s2

Many-sided progeny testing of bulls Several arguments were given for acquiring many-sided information of the transmitting ability of A. I. bulls. Many traits can be measured on phenotypes, but progeny testing is still required in many cases. The present Finnish routine includes milk and fat yields, fat content and live weight of daughters. Field trials have been performed with regard to several additional traits : Protein content and yield of milk. Ca. 4 600 samples were tested for clearing up the possibility of testing bulls on the basis of one sample per daughter for protein content. There were large differences between laboratories and between herds in protein content. Heritability was 0.25 , whence a repeatability of 0.7 should be obtained with 35 daughters and samples, while in monthly testing 170 samples are needed for the same accuracy. Beef-producing ability. In slaughterhouse data from 21 700 young animals, age accounted for 42 , herds 24 , areas 9, breeds 3 and birth months 1.5 p. 100 of the variance in carcass weight. Variation was wide even after correcting for age, sex, breed and area. Heritability was o. 16 and INTRODUCTION Many-sided information of the genetic quality of A. I. bulls is highly desirable a. o. for the following reasons : I . The potential influence of an individual bull is very great in the present era of frozen semen. ( 1 ) Presented in the Study Meeting of E. A. A. P., Genetic Commission Verona, Oct. 9 , 1972 . 2 . Both the efficiency and direction of genetic change depend on the information used as a basis for selection. 3 . It is necessary to investigate the consequences of one-sided selection and to predict the possible unpleasant correlated responses. 4 . It is valuable to prevent the gene losses on a rational way instead of at random, conserving all possible genes. Selection of e. g. bull sires could be performed more resolutely, if the top bulls are known not to possess negative traits. 5 . Different herd owners, areas or countries may have different goals and needs. Knowledge of all economically important traits of animals for sale is thus appreciated by importers. 6. Breeding goals may vary with time, because of changes in prices and other conditions. Semen deposited in semen banks should preferably have thorough ware descriptions. 7 . It is important to know the faults of a bull, in order not to use it for a cow or herd with similar faults. 8. The breeders and their advicers pay in any case attention to various positive or negative traits of daughters. In order to lessen the effect of biassed judgments and rumors based on individual cases it is important to acquire objective information. PERFORMANCE TEST OR PROGENY TEST With regard to several traits the book-keeping concerning semen could be based on phenotypic measurements of bulls. As examples of such traits may be mentioned : -growth rate (disadvantage : only one testing environment) : -adult weight (maintenance feed requirements) ; -feed conversion during growth ; -carcass quality and leanness (ultrasonics or after slaughter) ; -appetite and eating habits and preferences ; -male fertility (semen production traits, non-return rate) ; -frequency of difficult calvings and stillbirths ; -activity of various hormones, enzymes etc. in body fluids (studies concerning the usefulness of these need progeny tests). Information of relatives as e. g. dams (milk yield, milkability, udders, longevity, health, character) could also be utilized, but complementary information of progeny is in many cases desirable a. o. for the following reasons : i. Accurate evaluation needed because of economic importance of trait. 2 . Heritability too low to give accurate performance test. 3 . Trait measurable only on females. 4 . Trait measurable only on slaughtered animals. , 5 . Great possibilities for selection after progeny testing. 6. Progeny testing possible without prolonging generation interval too much. 7 . Clearing up the usefulness of performance tests presupposes progeny tests. For routine purposes it is necessary to weigh the costs of progeny testing against the value of the additional information obtained, but by searching for simplified methods for progeny testing this might become economically feasible and still sufficiently accurate for a greater number of traits than is usual to-day. The number of daughters included as well as the number and accuracy of measurements per daughter can be adjusted according to the economic importance and heritability of each trait. The main data sources available are : milk-recording, A. I. statistics, veterinary statistics, slaughter statistics, special collection by breeding advicers, and testing stations. PRESENT PROGENY TESTING ROUTINE IN FINLAND The present system is based on 12 months' records of milk and fat. Until 1972 the results were computed only for the official operational year (June I -May 31 ), but now rolling 12 -month records are also utilized. These make it possible to renew the tests every month if desired. A daughter is included as soon as she has milked 12 months after her ist calving and kept with until she has milked three years. The following traits are included at present : i. Relative milk deviation = average deviation of relative milk yield (p. 100 ) from the corresponding age average of the breed (corrected for month of calving and number of daughters). 2 . Relative fat yield deviation as for milk. 3 . Relative fat corrected ( 4 p. lO a) milk deviation as for milk. 4 . Fat content deviation from herd average (age-corrected). 5 . Simple index = (i) + 10 ( 4 ). 6. Live weight deviation = estimated live wt. -herd average (age-corrected, in kgs). Measuring the milk yield per unit time and including even 2 nd and 3 rd records make the test more many-sided than tests based on 305 -day lactation yields. It is especially safer with regard to fertility, since prolonged calving intervals tend to lower annual yields, while the 305 -day yields favour repeat breeders. Very little daughters are needed to compensate the loss in basic h 2 caused by our way of measuring the yield. A disadvantage in the live weight deviation is that the measures (body length and chest girth) are taken at varying stages of lactation. This harm is reduced with the increase of group size, and an r = 0 . 35 between 3 6 5 -day weight of bull in performance test and daughters' live weight deviation (n = r 3 6, Ayrshire) seems to indicate that the latter gives a useful picture of the adult size and maintenance requirements of daughters. Its value in evaluating beef production ability is somewhat doubtful, since the growth for beef should take place before the age of 1 -1 .5 yrs. In fact, one should require about i p. ioo unit increase in the relative milk deviation for each 10 kg increase in the live weight deviation. Considering the value of calf for beef production this requirement could be halved to 0 . 5 p. 100 units. In a sample of ca. 200 progeny-tested Ayrshire bulls the milk deviation increased by 0 . 2 p. 10 0 units per 10 kg increase in live weight. EXPERIMENTAL PROGENY TESTING FOR PROTEIN CONTENT Because of the increased actuality of including protein content among the grounds of milk payment and among the breeding goals, a field trial concerning about 4 6 0 o milk samples was performed in July-September, 1971 . The purpose was to clear up the possibilities of progeny testing bulls on the basis of one sample per daughter (ist-calvers) and to get actual progeny tests for a year class of bulls. The samples collected in July were analyzed with Promilk and Milkotester in laboratory I, and those from September with IRMA in laboratory II. The preservative in use was not always properly dissolved in July and many samples were kept in room temperature rather long time, and hence a proportion of samples was unsuitable or gave biassed results, especially for fat content. The useful part of data comprised ca. 4 . 000 cows. Information was available also of the test-day milk yield and of the fat content of the same day determined in the milk-recording with the Gerber-method. The relative importance of some external causes of variance is shown in table i. The figures are based on hierarchical analyses of variance, but the significances agree to great extent with the results of least-squares analysis. The great importance of laboratories is mainly due to the incomplete calibration of IRMA in laboratory II. The difference in protein content between laboratories was as high as o.q.q p. 100 units according to least-squares constants, even though the differences in time from calving were eliminated. It thus seems necessary to correct the probable differences between laboratories or analyzer types in handling milk composition data. The large herd components do not cause great problems in progeny testing in case the daughters are located in tens of herds. A part of the herd differences could be explained by herd averages for milk yield and fat content of the recording year. The regressions of all composition traits on these averages were significant in the I,S-analyses. Both fat determinations decreased by 0 . 05 p. 100 and protein content increased by 0 . 02 p. 100 per i o0o kg increase in herd average. P/P'-ratio increased by 1 . 4 p. 100 . An increase of herd fat p. 100 by I p. 100 was followed by o. 45 p. 100 (lab.) and 0.5 9 p. 100 (rec.) increase in fat content, o. y p. 100 increase in protein content and 4 . 7 p. ioo decrease in P/F-ratio. An increase of the time from calving to sampling by 100 days increased both fat contents by o.26 p. ioo and protein content by o.ig p. 100 while P/F-ratio decreased insignificantly. The differences between sires in the corrected (lab., areas, breeds, herd averages for milk and fat-p. 100 , time from calving) values were highly significant for all composition traits, and the h 2 -estimates were 0 . 25 , 0 . 32 , 0 . 3 6 and o.28, resp., for protein-p. 100 , lab.fat-p. 100 , rec.-fat-p. 100 and P/F-ratio. The estimates were similar for both laboratories, so the incomplete calibration of IRMA did not affect the accuracy. The lower h 2 for lab.-fat-p. 100 as compared to rec.-fat-p. 100 is explained by the improper dissolving and long conservation of some samples. A total of 144 bulls were tested on at least 10 daughters. The range within Ayrshire (114) was about i p. 1 00 unit for fat-p. 100 , 0 .7 p. 100 for protein and 20 p. 100 for P/F-ratio. The correlation between the two fat-p. 100 averages was 0 . 3 6, and the correlation of these with protein-p. 100 0 . 31 (lab) and o. 4 2 (rec.). On the basis of the h l -estimates a repeatability of 0 . 70 should be obtained with 35 daughters for protein p. roo, when there is only one sample per daughter. On the basis of io samples a year the corresponding number is y (h 2 = 0 . 5 ), i. e. 170 samples vs. 35 samples for the same accuracy. EXPERIENCES OF FIELD PROGENY TESTING FOR BEEF PRODUCTION Because of the availability of performance testing, progeny testing can be considered unnecessary for evaluation of bulls for beef producing ability. At the present state of knowledge, however, extensive progeny tests are needed merely for clearing up whether this conclusion is justified. It would be valuable to know ( I ) how the growth results obtained in an experimental environment are reflected in offspring reared in various practical conditions ( 2 ) whether the carcass quality of the offspring of well-grown bulls is satisfactory, and ( 3 ) how the progeny tests for milk and beef are correlated with each other. In case progeny testing could be made simple and cheap enough, it might be well-founded to check the results of performance test also regularly. The usefulness of slaughter house data, reported by milk-recorders on optical forms since 19 68, and the possibility of developing a method for progeny testing, have been studied on the basis of 21 700 animals. In over 9 8 p. 100 of the cases the age at slaughter was between 90 and 6 00 days, with rather normal variability and an average of 3 m days. Carcass weights varied from 10 to 2 6 0 kg, and the mean was 114 kg. 3!4 of the animals were males. An increase of one day in slaughter age increased carcass weights by 319 , 3 ro and 23 8 grams in the age groups z2o-26g, 2!o-38g and 39 0-5 6 9 days, respectively. Differences between age groups, herds, areas, breeds and birth months explained 42 p. 100, 24 p. 100, 9 p. 100, 3 p. 100 and 1 .5 p. 100 , resp., of the variance in carcass weights. The variation of these was surprisingly wide even after elimination of age, sex, breed and area differences : for 120 -3 6 5 days old animals the 9 -month weights varied from 40 to 1 8 0 kgs in Ayrshire and from 35 to 170 kg in Finncattle. For 3 66-56 9 days old animals the range at i 4 . 5 months was ca. '5 o kg. Thus the feeding of calves for beef is unsatisfactory in many herds. The range of herd means of at least 3 animals was I IO kg at 9 months, and the extreme differences between areas were 20 -30 kg in age-corrected weights. At the performance testing station the average weight of Ayrshire bulls at 3 6 5 days was 450 kg in 1971 , which means a carcass weight of over 230 kg. The C. V. of 27 o-day weights at the station has been less than 10 p. 100 , while that of corrected carcass weights of 120 -3 65-days old animals at 9 months was 21 p. 100 . The average h Z -estimates for corrected carcass weights were 1 6 p. 100 and 22 p. 100 , resp., for 120 -3 65-days old ( 11435 ) and 3 66-56 9 -days ( 3 8 13 ) old animals. Assuming an h 2 = 0 . 2 , a repeatability of 0 . 7 in progeny testing would be obtained with 44 offspring and b = 0 .5 with 20 . A total of 274 Ayvshive and I IO Finncattle bulls were tested on at least 10 offspring. The averages at 9 months varied from 8 3 to 115 kg in Ayvshive and from 77 to io6 kg in Finncattle. An attempt to utilize relative carcass weights (comparisons to herd means) decreased the number of tested bulls by 79 p. 100 and the group size of the remaining bulls by 71 p. 100 . Thus, the relative tests have to be abandoned, especially since the offspring of selected bulls compete with each other in the most interested herds. On the other hand, the confounding of feeding intensity and interest to top bulls lessens the value of the absolute tests. This bias is shown e. g. by the fact that the corrected carcass weight of 345 Chavolais X Ayrshire crosses was 23 p. 100 above Ayvshive mean, while the difference in our experiments has been only 9 p. 100 . During the last years we have tried to build a network of big specialized herds which are rearing calves mediated by slaughter houses and which are interested in collaboration. In these ca. 50 herds comparisons can be performed within herds, and the disturbing effect of correlation between feeding and genetic quality can be eliminated. AN INTERVIEW TRIAL CONCERNING MII,KABII,ITY The increased costs of human labor have made it is necessary to pay increasing attention to milkability, especially in large herds. In Finland, the interest in this trait has grown rather slowly because of the small herd sizes and since one cannot strive for extremely easy milking (risk of mastitis and milk leak). A hypothesis can also be made that the slowly milking daughter groups cannot compete with regard to relative milk yields in the hasty milking routine. This hypothesis may not, however, entirely hold in small herds. In order to get a large number of bulls progeny-tested cheaply and rapidly, an interview method was given a trial in i 97 2. The milk-recorders interviewed the herd managers concerning the ranking of the last three ist-calvers within the herds. By placing in each herd one cow to each possible rank ( 1 , 2 and 3 ), one hoped to avoid the usual disadvantage of subjective evaluation, that different judges utilize different parts of the scale and that only very limited part of scale is utilized. The judgment would be based on experiences during tens of milkings, and the computations would be simple, without various corrections. Collecting the data on optical forms made their handling easy and rapid. Useful data were obtained of 6 42 6 cows. The effect of time from parturition was only 0 . 25 p. 100 of variance. Heritability was ca. 0 . 0 8. The result can be considered encouraging, for the instructions did not go to the milk-recorders properly, due to short time and many intermediaries. A total of 1 6 2 bulls had at least 10 daughters. Ca. 20 p. 100 of the bulls differed more than 0 . 2 rank points from 2 . 0 to both directions, and the extremes were -f-0 . 55 and &mdash; 0 .6 4 . For those few bulls which had previously been tested with special equipment, the results were in reasonable agreement. More can be said after the results of a simultaneous trial on measuring milking time have been analyzed. INTERVIEW TRIAL ON CHARACTER, APPETITE AND ESTRUS SYMPTOMS In connection with the interview trial on milkability, similar information was collected on the character (calmness), appetite and estrus symptoms of firs-calvers. The h 2 -estimates were respectively i 3 p. 100 , 5 p. 100 and 2 p. 100 . Exactly the same bulls were tested as for milkability, and the variation was of course similar. PROGENY-TESTS FOR FERTILITY In the ig6o's extensive statistical analyses were performed by MeiJA!,A ( 19 6 4 , 19 66) on the genetic variation of various fertility traits in practice. The la2-estimates varied according to the measure used, the age of cow, the A. I. unit, year and the source of information, but the most common values were 1 -3 p. 100 . By utilizing repeated measurements on the same cows it was possible to judge individual cows with an accuracy of 6 p. 100 at 3 rd calving. On the basis of these estimates it was possible to calculate that a b = 0 . 7 in progeny tests should be obtained with 300 daugters for several fertility traits. This number is easily attained in A. I., but the basic A , tended to decline, when one came to larger progeny groups (selected sires), and hence it seemed difficult to reach a repeatability of o. 5 in practice. It is very important to eliminate the most important external factors, since even a small increase in h , at the vicinity of zero decreases considerably the requirements concerning number of daughters. For some fertility traits, as e. g. non-return rate, the test can be performed on heifers, so that the generation interval does not suffer. It is not yet clear, however, whether the heifer fertility is genetically related to cow fertility. There are some signs of that it is a question of partly different traits. It is likely that progeny test fpr fertility will be made a routine. For research purposes one tries to progeny test bulls for the frequency of multiple births, in order to see the possibilities of increasing the calf crop for beef production by genetic means. FREQUENCY OF STILLBORN CALVES L INDS T R6M ( 1970 ) has given the average frequency of stillborn calves for 43 Ayrshire bulls in 19 6 9/70 . The minimum number of births for adult dams was 200 and for heifer dams 100 . In the former case the frequencies varied from o.6 9 to 7 . 0 8 p. 100 and in the latter from i.i 3 to 6.5 2 p. 100 . The correlation between the two series of values was 0 . 17 . EXPERIMENTAL COLLECTION OF DISEASE DATA A trial was arranged in 1971/72 to collect information for progeny tests of the most important diseases. The veterinarians should have made a note of each treatment with a special code to a card in the herd, and the milk-recorders should have reported these notations on optical forms. The necessary information of descendance, age etc. would then have been found from the EDP-registers. The frequency of veterinarians carrying out the notations was rather low, however, and it appeared difficult to perform any statistical analyses. Some individual veterinarians are interested in the matter, and the work may be continued with them. In the case of promising results the circle could be expanded gradually, especially if those participating would regularly get surveys and reports based on the data. The experiences obtained in Germany (G RAVERT and S CHR6DFR , 1972 ) do not give very much promise. ABNORMAL CALVES !SO-CAI,I,!D LETHALS) In the first part of the ig6o's attempts were made to collect information of abnormal calves. Special forms were planned for reporting these, and the A. I. technicians were asked to send the calves to the Veterinary College for diagnosis. Ca. 200 cards were received annually, but it was difficult to draw any conclusions concerning lethal carriers. The symptoms were very vague and variable in most cases, and in the few cases they were clear one could not be sure about the paternage. Some cases were reported for most of the bulls. It became obvious that most of the abnormalities were caused by environmental factors. Since there are many more important traits and tasks in cattle breeding, the work on abnormalities has been almost discontinued. A COUPLE OF TRAITS CONSIDERED FOR PROGENY TESTING There has been some thought to measure the persistency of lactation on the basis of monthly milk recordings, available in EDP-registers. For example, the coefficient of variation of daily yields could be computed according to B RUUN ( 192 8). The importance of persistency has recently increased in Finland, because of the restrictions of oil concentrate importation and of the expansion of green silage feeding. Another trait of actual interest is the !aresislike leg fault which occurs in some families of bulls and which is claimed to occur also in females. SOME CORRELATIONS BETWEEN PROGENY TESTS FOR DIFFERENT TRAITS One of the main motives for many-sided progeny-tests has been to get possibilities for elucidating the possible consequences of one-sided breeding for milk. Preliminary estimates of correlations between some traits are shown in table 2 . In spite of the small numbers of bulls in most of the comparisons, some significant correlations were observed. The milk values, for example, were negatively correlated with fat-p. 100 and protein-p. 100 , but positively with carcass weight of progeny in the field. The last-mentioned correlation is obviously biassed, however, since the most advanced herds have been mostly interested in the top milk bulls. The routine fat-p. 100 test was naturally positively correlated with both trial tests and also with the protein-p. 100 , and the trial tests were correlated with each other. It is interesting to observe that the live weight deviation of daughters was clearly correlated with the carcass weight of young slaughter offspring (r = 0 . 31 ), even with the relative weight (o.2g). Its correlation with milk deviation was insignificant o.r 4 , but about similar values have been obtained from bigger materials. The positive correlation between protein-p. 100 and appetite is also interesting, although far-reaching conclusions cannot yet be drawn. The positive correlations among milkability, appetite and estrus symptoms may partly be caused by biassed judgment, since they were judged simultaneously on the same optical form. The herd manager may have been inclined to give the same rank for the same cow for several traits. In earlier studies of MAI JALA ( 19 66) various fertility traits were slightly negatively correlated with progeny tests for milk. The magnitude and even the sign of the correlation varied with the measures used for both milk and fertility, as well as with the age class in question. The antagonism is most obvious during the ist lactation, if milk yield is measured per year and not per lactation. CORRELATIONS OF PROGENY TESTS WITH PERFORMANCE TEST The necessity of progeny testing for various traits and the possibilities to intensive selection of bulls on the basis of performance test depend partly on whether there are antagonisms between traits which can be judged on the bull itself in a given test environment and those measured on progeny in varying environments. Preliminary results concerning these relationships are presented in figures i and 2 . In figure I the correlations are positive with almost no exception. The correlations of carcass weight show that the data from field are useful and that the growth results obtained in the specific test environment are reflected in the growth of progeny in varying conditions. Surprisingly, the calmness of daughters was as closely correlated with the growth rate of bull as was the carcass weight of offspring. A calm temperament may thus be an important presupposition for rapid growth and it appears to be an inheritable trait, even from sire to daughters. From figure 2 appears that the live weight measured on milking daughters is similarly correlated with a bull's own growth as the carcass weight of his young slaughter offspring ( 75 p. 100 males). The most surprising observation was the antagonism between bull's growth rate at testing station and his daughters' fat content. This could be explained so that families which prefer to convert energy to fat cannot compete successfully in growth. This negative association made also the correlation of milk -E-fat index with performance test lower than that of milk yield. Both of these correlations are partly caused by the bigger size of daughters of well-grown sires. NECESSITY TO CONSTRUCT AN INDEX Many-sided information of bulls and their offspring leads easily to harmful confusion in selection, thus slowering the genetic progress with regard to the main breeding goals. A proper weighting of different traits according to their economic importance, the accuracy of evaluation and interrelationships among them is therefore important. For the main selection decisions it is necessary to construct a selection index incorporating all the information to one figure, but the breeders have to know also the special good and bad points of each bull. Some traits which are positively correlated with the main traits can be left unmeasured and unconsidered, but their inclusion could in some cases also increase the judging accuracy and thus the rate of progress.

v3-fos

2019-04-25T13:10:20.712Z

1974-01-01T00:00:00.000Z

129990982

s2

Effectiveness of Paraquat As Influenced by Carrier Volume and Climatic Conditions This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1974 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. ·Effectiveness of Paraquat As Influenced by Carrier Volume and Climatic Conditions Charles A. Norwood, Research Agronomist Traditionally, herbicides applied with ground equipment have been combined with relatively· large (20 gallons per acre or more) volumes of water. This has been particularly true with postemergence herbicides in order to obtain complete coverage of the foliage for good control. One notable exception to this is Roundup. Roundup is more effective in a low volume of water, because it can be deactivated by salts present in the water. Paraquat, on the other hand, has usually been applied in 20 to 40 gallons per acre of water. Recently, research at Garden City and other locations demonstrated that Paraquat is also effective in low carrier volumes. The results of that research and also some additional research with Roundup are the subject of this report. Results The results of one experiment with Roundup, designed to illustrate the effects of carrier volume, are presented in Table 1. Note that the control of volunteer wheat decreased as the volume of water increased. Similar results are well documented and are the reason that low water volumes are recommended for applications of Roundup. Less well known is the influence of carrier volume on the effectiveness of Paraquat. Table 2 shows the control of Russian thistle and kochia with 16 oz of Paraquat, as affected by water volume. Maximum control of both Russian thistle and kochia occurred with 12 GPA, although statistically there was no difference between the 6 and 12 GPA rates (or between 6 and 36 GP A in the case of Russian thistle}. Control of Russian thistle was less with 36 GPA than with either 12 or 24 GPA. Control of kochia declined significantly at both 24 and 36 GPA. Conditions at application also can have an important effect on weed control. The effects of both carrier volume and climatic conditions on the control of volunteer wheat with 16 oz of Paraquat are shown in Table 3. The first application was made at 3 PM when the air temperature was 97°F, relative humidity 37%, and wind speed 10 to 12 mph. The second application was made at 7 AM the next morning when the air tern-· perature was 70°F, relative humidity 80%, and the wind less than 5 mph. Nearly 100% control was obtained with the 6 and 12 GPA water volume applied at 3 PM, while significantly less control was obtained with the 24 and 36 GPA volumes. Perfect control was obtained from all applications made at 7 AM. lr-nproved control with Roundup with lower volumes of water is due to less deactivation of the chemical by salts in the water. The reason for improved control with Paraquat in a low volume of water is less clear. It is unlikely that Paraquat is deactivated in the water. It is possible that better coverage is obtained with the finer droplets produced by the smaller spray tips used for low volumes of water. Finer spray droplets also can be obtained by increasing the pressure. However, if the pressure is increased above 35-40 psi with the preferred fan tips, excessive drift and loss of chemical will occur. A constant pressure of 30 psi was used in these experiments. Conclusions Improved weed control with Roundup due to low carrier volume is well known. Paraquat also can bf' used in low volumes of water. Under ideal conditions, the volume of water will have little, if any, effect on the performance of Paraquat. Under hot, relatively windy conditions, however, lower volumes of water may re· suit in increased effectiveness. In any event, this research indicates that low volumes of water will not diminish the effectiveness of Paraquat. Lower volumes of water also will require less frequent filling of the spray tank, resulting in savings of time and money. Trade names are used to Identify products. No endorsement of these products or criticism of similar products not mentioned is implied. This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu.

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2020-12-10T09:04:17.559Z

1974-02-01T00:00:00.000Z

237230180

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Toxic Metabolite Produced by Aspergillus wentii Mycelial extracts of an Aspergillus wentii strain grown on yeast-extract sucrose medium and initially isolated from country-cured ham were highly toxic when inoculated into chicken embryos or fed to mice. Moldy corn and rice were less toxic when fed to mice. Water extracts of moldy corn or rice or culture filtrates from yeast-extract sucrose medium were not toxic. Purification by thin-layer chromatography followed by crystallization yielded orange-red crystals that showed high toxicity and had a melting point of 285 to 286 C. Chloroform solutions of the crystals had absorption maxima at 270, 295, and 452 nm. The smallest amount of this component necessary to have zero hatchability of fertile eggs was 50 μg/egg. Mycelial extracts of an Aspergillus wentii strain grown on yeast-extract sucrose medium and initially isolated from country-cured ham were highly toxic when inoculated into chicken embryos or fed to mice. Moldy corn and rice were less toxic when fed to mice. Water extracts of moldy corn or rice or culture filtrates from yeast-extract sucrose medium were not toxic. Purification by thin-layer chromatography followed by crystallization yielded orange-red crystals that showed high toxicity and had a melting point of 285 to 286 C. Chloroform solutions of the crystals had absorption maxima at 270, 295, and 452 nm. The smallest amount of this component necessary to have zero hatchability of fertile eggs was 50 gg/egg. Toxicity of Aspergillus wentii to animals was first reported by Rabie et al. (5). They found that strains of A. wentii grown on corn caused toxic symptoms and death in ducklings, chickens, rabbits, and sheep. The most consistent lesions were found in the liver (4). Higher toxicity was reported for one strain at incubation temperatures of 20 to 25 C than at 30 C, and higher toxicity was reported for another strain at 25 C, compared with 20 and 30 C. Ether extracts of A. wentii isolated from corn and grown on rice were toxic to mice but not ducklings when administered intraperitoneally (6). Semeniuk et al. (9) found several strains of A. wentii to be lethal for mice when grown on wheat. In a survey reported by Scott (8) of toxigenic fungi isolated from cereal and legume products, none of five strains of A. wentii was toxic to ducklings. Recently, Chassis et al. (Proc. Ass. Southern Agr. Workers, Jacksonville, 1971.) reported that corn infected with A. wentii isolated from country-cured ham was toxic when fed to mice. The present investigation was undertaken to further study the toxicity of A. wentii isolated from country-cured ham and to purify and characterize the principal toxin(s) produced by this strain. MATERIALS AND METHODS A. wentii M-108 previously isolated from a countrycured ham by Leistner cooled, 106 spores harvested from CDA and 300 ml of sterile distilled water were added. The inoculated substrates were then incubated at 27 C for 2 months; after incubation, the moldy substrates were autoclaved for 1 h. In a second experiment, 105 spores were inoculated into 100 ml of sterilized yeast-extract sucrose (YES) medium (2% yeast extract, 20% sucrose) in 500-ml Erlenmeyer flasks, and the substrate was incubated at 27 C for 1 month. After autoclaving, mycelia were separated from the culture filtrate and air-dried. Chicken embryo inoculation (10) and mouse feeding were used to assay the toxicity of moldy substrates. For chicken embryo assays, dry moldy corn, rice, and mycelia were ground in a no. 3 Wiley mill. Fifty grams of mycelial powder from YES medium was extracted by using chloroform in a Soxhlet extractor. Also, 50 g of moldy corn or rice was similarly extracted. After 16 h of extraction, the extract was concentrated in a flash evaporator and diluted to 5 ml with chloroform. Various dilutions were also made for testing the level of toxicity. Groups of 20 fertile White Leghorn eggs were inoculated before incubation with 0.02 ml of water or chloroform extract or with culture filtrate from YES medium. Control groups of eggs were injected with chloroform or water, with chloroform or water extract of uninoculated corn and rice, or with sterilized uninoculated YES medium. For mouse feeding tests, ground moldy rice and corn and dry mycelia were mixed with ground Purina mouse chow (Ralston Purina Co., St. Louis) in different ratios. Chloroform extracts of mycelia and culture filtrates of YES medium were also mixed with mouse chow in different ratios. These mixtures were placed under vacuum to remove chloroform. Ten 2-week-old white Swiss mice were used in each treatment. Control groups were fed mixtures of uninoculated rice and corn with mouse chow in corresponding ratios. Water and feed were available ad libitum. The assay lasted 5 months. The criteria for positive response were weakness, recumbence, and death. 337 APPL. MICROBIOL. The chloroform extract of mycelia was separated by thin-layer chromatography (TLC) on Adsorbosil-1 (Applied Science Laboratories, State College, Pa.) with chloroform-acetone 95:5 (vol/vol) as the developing solvent. To obtain more material for assay of toxicity, 0.2 ml of chloroform extract was applied as a line on a TLC plate. After developing, the plate was divided into 13 bands according to color and fluorescence under ultraviolet (UV) light. Each band was removed and extracted with chloroform and acetone mixture. The bands with high R, values required more chloroform in the mixture for extraction. The extracts from five plates were concentrated to 1 ml and injected into 20 fertile eggs (0.02 ml/egg) for each assay of toxicity; duplicate tests were made. RESULTS AND DISCUSSION Of the 13 bands observed on TLC plates, band 6 (R. 0.57), which proved to be the most toxic, was collected from many plates and extracted with a mixture of chloroform and acetone 50:50 (vol/vol). Purity of this compound was checked with other solvent systems (chloroform-acetone, 50:50; toluene-ethyl acetate-formic acid, 6:4: 1; chloroform-ethanol, 95:5; benzene-ether, 5:2). When these plates were charred with sulfuric acid solution, only a single spot appeared. The extracted no. 6 bands were combined and concentrated, and the crystals formed were collected and dried under vacuum. The melting point of the crystals was determined by using a melting point apparatus. A Beckman DBG spectrophotometer was used to determine the absorption spectrum in the UV and visible ranges. Toxicity of this purified material was assayed by using the chicken embryo test. Table 1 shows the toxicity of different extracts to the chicken embryos. The culture filtrate of YES medium and water extracts of moldy corn and rice were not toxic to chicken embryos. Chloroform extracts of moldy corn were toxic to chicken embryos when introduced at full strength (0.02 ml) and slightly toxic at one-tenth that of the original concentrated extract. The chloroform extracts of moldy rice were moderately toxic, and no toxicity was observed at low concentration. Chloroform extracts of mycelia also were highly toxic. The extract from 10 mg of mycelia was the minimum amount necessary to obtain zero hatchability in 20 eggs. Table 2, moldy corn mixed with mouse chow (1:1) killed mice in 58 to 70 days, whereas a low ratio of moldy corn to chow (1:9) did not kill but only retarded growth. Moldy rice at 1: 1 and 1:9 ratios did not kill mice. Mycelia from YES medium was the most toxic substrate. A mycelia-mouse chow mixture of 1:9 killed all 10 mice within 4 to 6 days. On the There was no particular symptom before mice died except that their growth was retarded. They became weak, recumbent, and moribund. As shown in It is apparent that toxin(s) of this fungus is intracellular. Mixtures of uninoculated corn and mouse chow and uninoculated rice and chow were not toxic to mice. Since the chloroform extract of mycelia from YES medium showed the highest toxicity, isolation and purification of toxin(s) were attempted from this extract. The chloroform extract of mycelia was a dark-brown viscous liquid. Table 3 shows the relative toxicities of 13 bands from the TLC plates on chicken embryo hatchability, and band 6 apparently was the most toxic. Other bands such as 1, 2, 4, 7, 8, and 11 also show some toxicity. In this study, we only worked on band 6. The toxin appeared as a single orange-red band (Rf 0.57) on TLC plates. The toxin was soluble in chloroform, acetone, benzene, and ethyl acetate, insoluble in ethanol, and did not fluoresce under UV light. Crystallization from the chloroform-acetone 50:50 (vol/vol) yielded orange-red needleshaped crystals having a melting point at 285 to 286 C. The UV and visible spectra showed absorption maxima in chloroform at 270, 295, and 452 nm. The minimum amount of this toxin necessary to have zero hatchability was 50 ag/egg (Table 4). Chromatographic study showed this metabolite to differ from aflatoxins (B1, B2, G1, G2) and verruculogen (1,2,7).

v3-fos

2018-04-03T05:25:16.805Z

1974-08-01T00:00:00.000Z

42751894

s2

Evolution of Dimethylselenide from Soils Alcohols, carbonyl compounds, and fatty acids were formed in two glucose- amended soils incubated under argon, but dimethylselenide was evolved under argon only from one, a selenium-rich clay, after the addition of selenite and glucose. Substantial quantities of dimethylselenide were released from the four soils tested when they were incubated with glucose and selenite in air. No dimethylselenide was produced in the selenium-rich clay soil in air if it received glucose but no selenite. Fungi of several genera are able to form dimethylselenide in axenic culture, and species of Scopulariopsis, Penicillium, and Aspergillus have been demonstrated to be capable of synthesizing this product from inorganic selenium compounds (2,3). At least one coryneform bacterium also can convert inorganic selenium to dimethylselenide in vitro (J. W. Doran and M. Alexander, unpublished data). Nevertheless, apart from the demonstration of the evolution of trace quantities from samples of amended sewage (3), nothing is known about dimethylselenide formation in natural ecosystems. The potential for such an activity is suggested by a report that volatile selenium is released from soil apparently as a result of microbial action (1), but the identity of the compound (or compounds) was not determined. Selenium is present in many soils as selenate and selenite, and is probably present in organic compounds derived from plant tissues and microbial cells (6). In some regions, it is present in such high concentrations that plants accumulate the element to levels that are toxic to animals consuming the plants. On the other hand, the soils of many regions are deficient in this element, and, inasmuch as selenium is essential for livestock, its addition to the land is under consideration. Because volatilization would change the quantity in soil that might be available for assimilation by forage plants and also might lead to the presence of selenium in the atmosphere, possibly to account for its transport to remote areas such as Antarctica (8) and the ice sheets of Greenland (7), a study was initiated to assess whether this compound might be discharged from soil. MATERIALS AND METHODS Samples of four soils were used: a selenium-rich clay soil from South Dakota containing approximately 30 ppm of selenium, 2.5% organic matter, and with a pH of 7.2; Honeoye silt loam from New York, with a pH of 5.5 and 5.6% organic matter; Croghan loamy sand from New York, with a pH of 5.9 and 4.6% organic matter; and a sandy loam with a pH of 5.9 and 0.5% organic matter from the Sonoran Desert near Tucson, Ariz. The soils were air-dried and passed through a 2-mm sieve prior to use. The Croghan loamy sand had been stored air dry for approximately 15 years, and the other soils had been stored for periods ranging from 6 months to 2 years. A 10-g sample of soil was placed in a 50-ml glass bottle equipped with an inlet and an outlet tube. The soil was amended with water only or with a solution containing 100 mg of glucose or 100 mg of glucose and 10 mg of NaSeO, in sufficient water to bring the soil to field capacity. The inlet tube of the sample bottle was connected to a gas manifold, and the outlet was connected to a stainless steel tube 15 cm long by 3 mm outer diameter. The steel tube contained Porapak QS (100 to 120 mesh). A stream of high-purity grade argon or pure-grade air (Union Carbide, Linde Division) was passed through the sample bottles to sweep the volatile compounds present in the head space into the Porapak trap, where they were retained. The flow rate was regulated at 1 to 2 ml/min by means of a needle valve placed between the sample bottle and the Porapak trap. The needle valve and a clamp placed between the sample bottle and gas manifold allowed the sample to be isolated when the trap was removed for analysis. The volatile metabolites retained in the trap were analyzed with a gas chromatograph-mass spectrometer, Perkin-Elmer model 270, at regular intervals. The trap was inserted into the injector port of the gas chromatograph and connected to the chromatographic column. The injector port was heated at 250 C to inject the contents of the trap into the chromatographic column (1.83 m by 3 mm outer diameter), which contained Chromosorb 101 (100 to 120 mesh). The column temperature was maintained at 25 C for 4 248 EVOLUTION OF DIMETHYLSELENIDE FROM SOILS min, and then it was programmed to 100 C at a rate of 32 C/min and from 100 to 250 C at a rate of 6.5 C/min. The mass spectrum obtained for each compound was compared with the mass spectra of authentic compounds for the purposes of identification. RESULTS AND DISCUSSION Soils receiving water but no other amendment did not produce detectable amounts of volatile compounds under either air or argon. When the seleniferous clay soil was amended with glucose or glucose and sodium selenite and incubated under argon, however, ethanol, n-propanol, nbutanol, acetone, methyl ethyl ketone, methyl propyl ketone, acetic acid, and butyric acid were found at various times during a 48-day incubation period. In addition to these compounds, traces ( <2 ug) of dimethylselenide were found when sodium selenite was included in the amendment. Similar experiments with the silt loam yielded ethanol, n-and isopropanol, n-butanol, acetone, methyl ethyl ketone, ethyl acetate, ethyl butyrate, and butyl butyrate. Dimethylselenide was not detected, and the only significant difference in products between the silt loam receiving glucose alone and the same soil receiving glucose and selenite was the generation of n-hexanol when the selenium salt was added. All four soils amended with glucose and NaSeO and incubated under a stream of flowing air evolved dimethylselenide. The quantities of this metabolite evolved from the four soils during a 48-day incubation period are shown in Table 1. The initial rate of evolution was more rapid in the silt loam and the seleniferous clay than in the other soils, but a considerable amount of dimethylselenide appeared in the sandy loam late in the incubation period. Little evolution was evident from the loamy sand. The amount of selenium recovered as dimethylselenide was approximately 2% of that added with the seleniferous clay, sandy loam, and silt loam, and the recovery was about 0.3% with the loamy sand. Ethanol and acetone were also found in the headspace over the silt loam, seleniferous clay, and loamy sand during the first 15 days, but only dimethylselenide was detected in the headspace thereafter. No volatile organic compounds other than dimethylselenide were detected in the sandy loam. When the seleniumcontaining South Dakota clay was amended with only glucose and incubated in air, ethanol, acetone, iso-propanol, and methyl ethyl ketone but no dimethylselenide were detected in the first 7 days, and no volatile compounds were evident in the next 53 days. The results suggest that the microbial methylation of selenium is potentially widespread. This activity was found in the seleniferous soil and also in other soils when a readily available carbon source and sodium selenite were added. Because the seleniferous clay did not evolve the alkyl selenium product when amended with glucose only, the evolution must require that soils contain the element in a suitable concentration or in a form which is readily utilized by the microorganisms responsible for methylation. Volatile organic selenium compounds other than dimethylselenide were not detected by the procedures employed, yet they may be generated under certain circumstances. The ecological significance of the finding that a volatile methylated selenium compound can be formed in soil is not clear. In this regard, it is noteworthy that selenium is found in the atmosphere above the geographic South Pole (8) and in the ice sheets of Greenland (7), and it is plausible to suggest that the element reaches these remote sites as a result of the discharge of dimethylselenide from soils in the tropics or temperate zone. Indeed, a parallel may be drawn with dimethylsulfide, which has recently been proposed to be the major volatile carrier of sulfur in the natural sulfur cycle (4). If dimethylselenide is, in fact, a common metabolite evolved from natural ecosystems, it would also be of great importance to learn more of its toxicity, especially in view of the known hazards of methylmercury and trimethylarsine. McConnell and Portman (5) reported that dimethylselenide had a low mammalian toxicity, the median lethal dose to mice and rats being greater than 1.0 g/kg when given intraperitoneally, but of significance in the context of the present investigation would be the toxicity of inhaled dimethylselenide. Furthermore, the identities of the organisms responsible for the formation of dimethylselenide in soil have yet to be determined, although it is known that fungi of diverse genera are able to bring VOL. 28, 1974 249 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from about selenium methylation in culture media. It is clear, therefore, that additional studies of the ecological significance, the microbial formation, and possibly the toxicology of dimethylselenide are warranted. ACKNOWLEDGMENT This investigation was supported by grant NGR 33-010-127 from the National Aeronautics and Space Administration. We thank A. N. MacGregor and 0. E. Olson for providing us with soil samples and Scott Smith for his able technical assistance. @

v3-fos

2020-12-10T09:04:20.804Z

1974-12-01T00:00:00.000Z

237233880

s2

Microbial Metabolism and Dynamic Changes in the Electrical Conductivity of Soil Solutions: a Method for Detecting Extraterrestrial Life The addition of 0.5% glucose solutions to 12 different air-dried soils always resulted in increased electrical conductivity and water-soluble Ca and Mg in the soil solutions. The kinetics and magnitude of these changes for at least two and usually all three of these parameters over a 14-day period were clearly distinguishable from the changes in heat-sterilized controls or unsterilized controls without added glucose. In general, maximal values were achieved more rapidly under aerobic than anaerobic incubation. Some soils (less than half) also showed significant increases in water-soluble Na or K when compared with the controls. The 12 different soils studied represented four general soil groups: I, leached acid upland soils; II, saline alkaline soils; III, nonsaline neutral soils; and IV, high organic soils. Viable counts ranged from 104 to 107 per cm3 of air-dried soil. Glucose metabolism by the indigenous soil microbiota was always accompanied by a significant decrease in the pH of soil solutions, but not necessarily by an increase in the viable count. The feasibility of using electrical conductivity and water-soluble Ca and Mg measurements to detect metabolic activity, either alone or in conjunction with other life detection techniques, is discussed. The addition of 0.5% glucose solutions to 12 different air-dried soils always resulted in increased electrical conductivity and water-soluble Ca and Mg in the soil solutions. The kinetics and magnitude of these changes for at least two and usually all three of these parameters over a 14-day period were clearly distinguishable from the changes in heat-sterilized controls or unsterilized controls without added glucose. In general, maximal values were achieved more rapidly under aerobic than anaerobic incubation. Some soils (less than half) also showed significant increases in water-soluble Na or K when compared with the controls. The 12 different soils studied represented four general soil groups: I, leached acid upland soils; II, saline alkaline soils; III, nonsaline neutral soils; and IV, high organic soils. Viable counts ranged from 104 to 107 per cm' of air-dried soil. Glucose metabolism by the indigenous soil microbiota was always accompanied by a significant decrease in the pH of soil solutions, but not necessarily by an increase in the viable count. The feasibility of using electrical conductivity and water-soluble Ca and Mg measurements to detect metabolic activity, either alone or in conjunction with other life detection techniques, is discussed. Any analysis of the problem of detecting extraterrestrial life resolves itself ultimately into the question of how one defines a living system. Young et al. (16) considered the problem from the biologist's point of view and concluded that living systems would have in common the attributes of (i) a carbon-based macromolecular chemical composition; (ii) the ability to grow and reproduce; and (iii) the ability to utilize materials and energy through the process of metabolism. Although many life detection experiments have been proposed, all fall within one or more of these categories (reviewed in 2, 6,8,10,15,16). Four experiments bearing on the detection of life have been selected by the National Aeronautics and Space Administration for the 1975 Viking mission to Mars (4,9,12). In one experiment Martian soil will be analyzed for volatile and pyrolyzed organic compounds by gas chromatography-mass spectrometry (1). The remaining three experiments are designed to detect metabolic activity in Martian soil. One experiment measures photosynthetic and dark fixation of 'IC-labeled CO2 and CO under I Dedicated to the late Wolf Vishniac, one of the pioneers of exobiology, who died in the Antarctic in December 1973. relatively dry conditions (3); the second measures the release of "4CO2 from a dilute aqueous solution of l4C-labeled organic compounds added to soil (5); the third measures the production or consumption of H2, N2, 02, CH4, and CO2 by soil incubated with a complex, aqueous, organic medium (7). From an ecological point of view, all living systems interact with the gas, liquid, and solid phases of the environment, but the three Viking metabolic experiments (3, 5, 7) measure only interaction with the gas phase, as does the unified approach proposed by Radmer and Kok (11) for subsequent Viking missions. If one assumes that water is as essential for life on Mars as on Earth, then Martian metabolic processes should also interact with the aqueous phase of the environment. As nutrients are converted into lower-molecular-weight metabolic intermediates and organic or mineralized end products, there should be a net increase in the number of ionic and electrically charged organic and inorganic molecules in solution and a corresponding increase in the electrical conductivity (decrease in impedance) of the aqueous phase of the environment. Dynamic E42, 1974). An automated system for impedance measurements in bacteriology has been described (P. Cady and S. W. Dufour, Abstr. Annu. Meet. Amer. Soc. Microbiol., Abstr. E43, 1974). This paper reports the results of studies on dynamic changes in the electrical conductivity and other parameters of soil solutions as a function of metabolism by the indigenous microbiota and on the feasibility of their use as metabolic probes for detecting extraterrestrial life. MATERIALS AND METHODS Aerobic studies. Thirty cubic centimeters of soil and 80 ml of liquid in 300-ml Erlenmeyer flasks were incubated in the dark at 30 C without agitation. Ten-milliliter portions were removed and centrifuged in 15-ml Corex centrifuge tubes for 10 min at 10,000 x g. The supernatant fluid was filtered through a 0.22-agm membrane filter, and the filtrate was used for pH and electrical conductivity measurements as well as for analyses of water-soluble Na, K, Ca, and Mg. Three different flasks were used for each soil. One contained 30 cm' of soil and 80 ml of filter-sterilized 0.5% glucose solution. A second contained 30 cm8 of soil and 80 ml of sterile water. The third contained 30 cm' of dry heat-sterilized soil (160 to 170 C, 94 to 96 h) and 80 ml of sterile 0.5% glucose solution. The soils were dispensed by equal volumes rather than equal weights to conform more closely to actual practice on the Viking Mars mission (4). Anaerobic studies. Three cubic centimeters of soil and 8 ml of liquid were added to 15-ml Corex centrifuge tubes capped with Morton stainless steel closures. The tubes were incubated at 30 C in the dark under H, + CO, in GasPak system anaerobic containers (Bioquest). Three sets of tubes were used for each soil studied. One set contained unsterilized soil plus filter-sterilized 0.5% glucose solution; one contained unsterilized soil and sterile water; and one contained dry heat-sterilized soil (160 to 170 C, 96 h) and filter-sterilized 0.5% glucose solution. One tube was taken from each set on days when measurements were to be made. One milliliter was removed from each tube and incubated in thioglycolate broth (Difco) for viability and sterility checks. The remainder of the tube contents was then centrifuged and treated as for the aerobic studies. Viable counts. Aerobic plate counts were made by taking 1-ml portions from the aerobic flasks, making decimal dilutions in dilute Trypticase soy broth (BBL, 1 g/liter), spreading 0.1 ml of the decimal dilutions on the surface of dilute Trypticase soy agar plates in triplicate, and incubating the plates at 30 C for 7 days. Viable aerobic counts are expressed as colony-forming units per ml of soil-liquid mixture, except for the counts in Table 1, which are expressed as colony-forming units per cubic centimeter of airdried soil. Soils. Twelve different soils were studied. All were provided by the University of California, Davis, except Death Valley iS and Yolo, which were collected by E. L. Merek of our laboratory. All soils were collected from the upper 30 cm, air-dried, sieved through 0.25-inch (about 0.8 cm) mesh, and mixed before being stored in glass jars. The soils are divided into four different groups: leached, acid upland soils (group I), saline alkaline soils (group II), nonsaline neutral soils (group III), and high organic soils (group IV). Descriptions and analyses for each soil are given in Tables 1 and 2. Analytical. The electrical conductivity of the soil solutions was measured at ambient temperature (22 1 C) by using a Beckman Instruments conductivity cell with platinum electrodes (no. GlOY184) in conjunction with a model RC16B2 conductivity bridge from Industrial Instruments, Inc. Water-soluble Na, K, Ca, and Mg were measured in appropriate dilutions with a Perkin-Elmer model 303 atomic absorption spectrometer. RESULTS Both aerobic and anaerobic experiments were conducted and gave reproducible results when repeated. The results of the anaerobic experiments are emphasized because oxygen in the Martian atmosphere is either absent or below the level of detection. Under anaerobic incubation, significant changes occurred in the electrical conductivity of soil solutions from group I soils when supplemented with glucose. These soil solutions, with low initial electrical conductivity (Table 2), showed much greater changes with time in electrical conductivity than control solutions from sterile soil supplemented with glucose or unsterilized soil with only water added. Representative results with Siskiyou soil are illustrated in Fig. 1. Solutions from group III and IV soils, with somewhat higher initial electrical conductivity, also showed significant changes in electrical conductivity when compared with the controls. Typical results with Ramona (group III) and Staten (group IV) soils are shown in Fig. 2 and 3, respectively. A more severe challenge to the use of electrical conductivity measurements to detect metabolic activity was presented by the saline alkaline soils from group II, with sizeable quantities of extractable cations and relatively high initial electrical conductivity (Table 2). Metabolic activity was easily detectable with Waukena and Death Valley 1S soils within 7 days (Fig. 4). With Holtville, detection of metabolic activity was more equivocal. It took 11 days before the electrical conductivity of the glucose-supplemented solution exceeded that of the controls by approximately 1,000 Amho/cm. When incubation was aerobic rather than anaerobic, changes in the electrical conductivity of glucose-supplemented solutions from all the soils, including Holtville (Fig. 5), were clearly distinguishable from the changes observed in the controls. Changes in the electrical conductivity of soil solutions resulting from the metabolism of glucose by the indigenous microbiota were also accompanied by dynamic changes in the watersoluble Ca and Mg for all 12 soils, whether incubated anaerobically or aerobically. The one exception was Waukena, incubated aerobically, which did not show a comparable increase in water-soluble Mg. Representative results of only the anaerobic experiments are shown in Fig. 6 for Ca with Ramona soil (group III) and Fig. 7 for Mg with Death Valley 1S soil (group II). Comparable results were not always observed for water-soluble Na and K under aerobic and anaerobic incubation. Less than half the 12 soils tested yielded solutions that showed significant increases in these elements when compared with the controls. Metabolism of glucose by the indigenous microflora always resulted in a decrease with time of from 1 to 2 pH units compared with the controls, regardless of the initial pH, the soil tested, or whether incubation was aerobic or anaerobic. This is illustrated in Fig. 8 12 I because as a nonionic compound it had no effect TIME, days as such on the zero-time electrical conductivity population of a given soil. Our results show that metabolism of glucose was easily detected in all the soils tested, under anaerobic or aerobic incubation, by measuring the kinetics and extent of the changes in electrical conductivity, water-soluble Ca and Mg, and pH. The kinetics of the increase in water-soluble Ca and Mg ( Fig. 6 and 7) and electrical conductivity as a result of metabolic activity generally paralleled one another but appeared to reach E maximal values more rapidly under aerobic 8 than anaerobic incubation. In general, more Ca E than Mg was released to solution and probably > was related to the relative amounts of extracta-> ble Ca and Mg in the soil. All the soils except A Aiken contained a sizable excess of extractable a Ca over Mg (Table 2).°S terilization of soil for use as a control de-< serves some comment. We sterilized the soils by = dry heat in air. Under these conditions, the 2 indigenous organic matter undoubtedly was W oxidized as evidenced by the yellow-to-amber color of the soil solutions (solutions from unsterilized soils remained colorless). The intensity of the color seemed proportional to the quantity of organic carbon in the soil ( Table 2). The soluble oxidized organic matter also led to increases in the initial electrical conductivity in most of the soils (Fig. 1-5), some decreases in the initial pH (Fig. 8), and an increase in initial water-soluble Ca (Fig. 6). These effects did not interfere with the subsequent kinetics and our ability to clearly distinguish microbial metabolism in unsterilized soils amended with glucose whose viable counts ranged from 10O to 107 per cm3 of soil (Table 2). We do not know whether heat-sterilized controls would interfere with the detection of metabolic activity in soils harboring smaller populations. If so, then eliminating heat sterilization of soil as a control might be considered since soils with only water added appear to be as good, if not better, controls. There are a number of advantages to measuring metabolism by electrical conductivity from the standpoint of a life detection experiment on Mars. (i) The method is nonspecific; i.e., it does not depend on the detection of a specific metabolite but responds to increases or decreases in the concentration of all electrically charged molecules and ions in solution, both organic and inorganic. (ii) The measurements are nondestructive and could be made continuously or at any convenient time interval, thereby enabling the kinetics of the process to be followed. (iii) Measurements can be made over a wide dynamic range, at least three decades in the 12 soils tested despite low or relatively high initial values (Table 1 and Fig. 1-5). (iv) No new technology is necessary. It should be relatively simple to insert electrodes in a cuvette assembly containing Martian regolith and an aqueous solution or to incorporate the electrodes as an integral part of the cuvette itself, as already demonstrated in a commercial instrument dynamic changes in water-soluble Ca and Mg. Ion-specific electrodes are available for measuring water-soluble Ca and Mg, they can make continuous, nondestructive measurements over a wide dynamic range, and, even in the absence of life, they would almost certainly yield some information on the geochemical nature of the Martin regolith. However, ion-specific Ca and Mg electrodes are sensitive to only the ionic forms of these elements, (Bactomatic, Inc., Palo Alto, Calif.). (v) A measurement of some kind is always obtained. In the absence of life or organic matter in the sample, at least some indication of the solubility and ionic nature of the Martian regolith would be obtained. Information of this kind would aid geochemists and cosmologists in understanding the evolution of the planet and its aqueous history. Many, but not all, of the advantages noted above for electrical conductivity measurements also hold for measuring metabolic activity by ble Ca and Mg was released in ionic form as opposed to undissociated inorganic or organic salts or chelates. Alternatively, it may be possible to adapt the X-ray fluorescence spectrometer on the Viking Mars lander (14) for use in inorganic analyses of water-soluble inorganic matter released in metabolic experiments. We conclude that measuring metabolic activity in soil solutions by means of dynamic changes in (i) the electrical conductivity, (ii) water-soluble Ca, or (iii) water-soluble Mg are feasible life detection methods. They represent alternatives to measuring metabolic activity in the gas phase for life detection (3-5, 7, 11) and can stand on their own as individual life detection experiments. They also demonstrate that the results of any life detection experiment, whether positive or negative, gain credibility in proportion to the number of different independent measurements made on the same sample. If any of the 12 soils we tested had been extraterrestrial in origin, the positive results obtained simultaneously for two or more of the three different parameters (electrical conductivity, water-soluble Ca, and water-soluble Mg) would lend enormous confidence to the conclusion that life was present. The nondestructive nature of electrical conductivity and water-soluble Ca or Mg measurements also commends one or more of these for use in conjunction with other life detection techniques, especially those that are destructive of sample or are restricted to only one measurement, e.g., analyses for proteins, nucleic acids, Dor L-amino acids, specific enzymes, etc. The experimenter in such cases must decide, often arbitrarily, when to perform his experiment. If he could choose the optimal time to perform his experiment based on dynamic evidence of metabolic activity, his chances of achieving a significant result would be greatly enhanced.

v3-fos

2018-04-03T05:03:29.572Z

2009-02-12T00:00:00.000Z

41414097

s2

Silverudd‘s Multiple Cock Shift System (SMCSS) Tests of Reciprocal recurrent selection (RRS) type actualize the need of continuous shifts of cocks in breeding pens without losing the pedigree accuracy for female chicks (RRS is not concerned with male chicks) hatched from eggs laid the first time (about 3 weeks) after a shift. Silverudd‘s multiple cock shift system (SMCSS) is based on definite colour genotypes of the strains. The variations of colour of chicks after different cocks will indicate the male progenitor of all female chicks (in many cases also of male chicks). When shifting cocks in the pens, SMCSS will safeguard full pedigree for all female chicks, even when several cocks are kept in one pen, provided the shifting is done in accordance with the rules governing SMCSS. Starting with only 4 groups, 144 sexable combinations can be made for as many RRS-tests in less than a one year. SMCSS may very well be utilized in such a way that, at the same time, complete sexing automation is achieved. Through the medium of SMCSS entirely new possibili- ties have been created for a time-saving and more comprehensive RRS and for obtaining a combination of strain breeding and RRS. 1. Reasons for experiments with crossings Some twenty years ago, when studying the genetic background for producing the so-called autosexing breeds (the first one produced by PUNNETT and PEASE 1930), my interest was soon aroused to attempt producing an autosexing type of White Leghorn. It is true that something had earlier been published about a technique of conveying WL genes to an autosexing breed and also something of utilizing WL (PEASE 1951) for the actual production of an autosexing breed. I found that, for producing autosexing breeds, I required a technique that allowed for more than 90 % WL-genes at the outset. My attention had been drawn to colour mutations, suppressed by dominant white (I) in White Leghorn. In 1956, an experiment was started with a double end in view, i.e. to produce an "autosexing White Leghorn" with predominant WL-blood, and, also, to get an idea of the colour mutations of the laboratory fowls. In order to eliminate dominant white from WL I had to cross in another breed of some kind. Through crossing with Brown Leghorn and recrossing to White Leghorn, dominant white was, by way of selection, eliminated from the stock. The colour factor for wild-type (e+) with Brown Leghorn, producing striped down, was cultivated in isolation within the stock, and, as expected, produced sexability in combination with barring, B (PUNNETT 1940). Out of the experiment emerged Fiftyfwe Flowery (FF), 1958(SILVERUDD 1967. The experiment also indicated that, in WL, there exist colour genes, which, in combination with B, can give autosexing. Thus it had been proved that, in order to change it to an autosexing breed, it will not be necessary to add a single gene to WL from another breed. In 1960, the gene for silver (S) was bred into FF to make FF ideal as hen strain, with a view to produce sex-linked crossbreeds. Silver strains of FF are called SFF and gold strains GFF. Having been bred for more than ten generations since 1960 without the supply of new blood, FF is now very homogeneous and is raised in Sweden for commercial purpose. While producing FF the desirability was kept in mind of retaining a number of genes from old Swedish strains that had been raised in Sweden for several decades. The basic condition for an ideal sexing automation has been studied (especially with regard to WL) in connection with many different crosses, comparing their particular genotypes and phenotypes (SILVERUDD 1968). This gave impulses for studies of the genetics of autosexing in relation to the great problems of breeding techniques, caused by the fact that, after shifting cocks in pens, pedigree accuracy on the paternal side is lost for a period of about three weeks. I found that, in shifting cocks in pens, the colour genes may be adapted in a fascinating way, due to the fact that by the colour of the female chicks (wether dayold or feathered) it will be indicated without fail, which cock is the actual progenitor, in each case. Silverudd's multiple cock shift system (SMCSS) admits of continuous changes of cocks without jeopardizing the pedigree accuracy (SILVERUDD 1970). The fact that, (as hen strain) SFF has a genotype that makes the breed more suitable for SMCSS than practically all other breeds has had as a result that, as far as hen strains are concerned, the interest in SMCSS has especially been coupled with SFF. Designations of genes A great variety of genes may have reference to SMCSS. Here below are specified primarily the genes included in the tables, and then those that are mentioned in the text only. Genes with Z-loci B = barred b+ = unbarred S =silver s+ =gold K = slow feathering k+ =rapid feathering Id = inhibitor of dermal melanin s a l = albinism Autosomal genes mo = mottled (with Ancona and others) Mo+ = non-mottled nj =the particular colour factor with Norwegian Data concerning the above-mentioned genes The gene designations are in many cases not identical with those used in "Genetics of the Fowl" (HUTT 1949). In its place the more up-to-date and accurate gene symbology, used by Smyth, Somes, Moore (cf. Literature cited) and others has been adopted. B may be called "the autosexing gene", because it is imperative in all autosexing breeds. However, it is only in a combination with, for instance, striped or mottled down, that autosexing can be achieved to 100 %. Barred Plymouth Rock has B, but, as carrying extended black (E), the breed is not 100% sexable. White Leghorn always has B. S, like B, is one of the most valuable genes, being highly important for sexing automation and SMCSS. Excepting the black element in them, breeds such as NH and RIR become white, if bred with S. Light Sussex is one of the best known silver breeds. K is not a popular gene, 'as late feathering tends to increase the frequency of feather picking and cannibalism. It may be possible, merely by the use of K, to grade WL-chicks with a 100 7,; accuracy (k+k+ cocks X K-hens). WL-hens may also produce double sex-linked combinations (SLVERUDD 1968 pedigree should be expected and be evident at an early stage, preferably already by the down colouring. When cocks are shifted, or when several cocks are kept in a pedigree pen, there will be many possibility of pedigree accuracy, so that no pedigree of interest is lost. Pedigree hatching. Presupposes trap-nesting, so that the eggs from a particular hen can be marked with the number of this hen and be hatched separately. The chicks will be wing-marked as day-olds, and the numbers registered, so as to keep track of pedigree without any break. Reciprocal recurrent selection (RRS). The purpose of RRS is to develop strains which, when crossed, yield progeny superior to either parental strain. "Selection is based not on the performance of the individuals within the strain but upon their ability to combine well or nick to produce the maximum of hybrid vigour. In order that the strains or breeds remain more or less flexible for selection purposes, inbreeding is avoided. Avoiding inbreeding also avoids, of course, the fixing of undesirable characters and the decrease in viability usually associated with inbreeding." (JULL 1960.) It may be mentioned that modifications of RRS exists (FALCONER 1964). Strain pedigree. A simpler form of pedigree, when registration is restricted to the paternal side. Individual control through trap-nesting, will then not be required within the stock. In many cases, the procedure of marking the wings may then be replaced by puncturing the skin between the toes according to a particular system. If hens in a group should have the same parents, strain pedigree may often be required. Cock group. Consists of cocks that have been coordinated in a group in such a way that, with the help of SMCSS, the group may be interchanged with another particular cock group, without losing any pedigree of interest on the paternal side. 2-inbreeding. Implies planned achievement of homozygosity for several sex-linked genes, resulting in a type of inbreeding that will effect the cocks, but not the hens, the latter being hemizygous for Z-genes and hence immune to defects from 2-inbreeding. Autosexing. Implies that a breed is sexable in itself. Legbar, Cambar, Rhodebar, Fiftylive Flowery, Norwegian Jaerhons, among others, are autosexing breeds. The first autosexing breed, Cambar, was produced in 1930 by PUNNETT and PEASE. Also crosses between autosexing breeds are to be regarded as autosexing. Sex-linked sexing. Sex-linked sexing involves a sexability by crossing two (or more) breeds or varieties, that are not sexable in themselves (or by crossing non-autosexing X autosexing). RIRX LS, N H X LS, BrL X LS, BrL X BR and BIL X BR are typical examples. There are also triple crossbreeds belonging to this category, e.g. BrL X (LS X NH), RIR X (LS X NH), RIR X (BR X AuO) and BIL X (SFF X NJ). Also combinations between cocks of rapid-feathering strains and hens of late-feathering strains are included in this category (feather sexing). The concept autosexing is often used of combinations that are sex-linked only. This practice creates confusion and should, therefore, be abolished. 4-Numerical codes for sexing methods In order to bring about consistency regarding various concepts in the field of sexing automation, I have adopted the following numerical code (SILVERUDD 1970): Not sexable, or only partly so Sexable, but not 100 % (at least 80 %) Sex-linked sexing through mating k+k+ (cock) X K -(hen) 0- Sex-linked sexing through combination of gold X silver or non-barred X barred. Thus s+s+ X Sand b+b+ X B -, respectively Double sex-linking through 2 + 1 Autosexing Autosexing and sex-linked sexing through 4 + 1 Autosexing and sex-linked sexing through combination of 4+2. The ideal form of sexing automation Triple sex-linking through 4 + 2 + 1 One of the advantages of this system is that of admitting tabular statements of hundreds of different crossings in a clear and concentrated form (SILVERUDD 1970). It is to be noted that, in many cases, 4 + 2 may produce combinations with such complete sexability that even one million dayold chicks may be sexed for sale without the risk of a single cock slipping through. When crossing two strains of WL (gold cock strain X silver hen strain) that have been applied for 4 + 2, sexability will be achieved to 100 % much earlier than is the case with the pure strains. Strains A. Hen strains Table 1 presupposes use of the silver variety of Fiftyfive Flowery (SFF) (SILVERUDD 1967) as hen strain(s). I n the four breeding-pens, SFF strains may be used. Having a genotype that suits SMCSS better than does almost any other breed, SFF has been selected as hen strain for the demonstration of SMCSS. SFF, as hen strain, will meet all reasonable demands on a perfect breed with regard to colour genotype. FF is the first Swedish poultry breed. It appeared in 1958 (in gold, a little later in silver). Fiftyfive has gone into the name of the breed, because, in 1955, the first crossbred chicks were hatched, later to be used for experiments started in 1956. Flowery refers to the white mottled markings of the hens, caused by mo (SILVERUDD 1967). For the production of FF, old Swedish strains were utilized, primarily some WL-strains from Overlida Poultry Farm, but also a WL-strain from Hirsbacks Poultry Farm, and one of Brown Leghorn. The genes B, mo, and e+ function in a threefold concurrence and harmony that is striking, giving to the breed its unmistakable and characteristic traits. FF has B and mo from WL, and e+ from Brown L. The combination of B, mo and e+ is extremely successful. When Ancobar was produced (LAMOREUX 1941), B was combined with mo and E of Ancona. But e+ has proved to be far superior to E in the combination of mo and B. Both with respect to autosexing accuracy, possibilities of a 100 % sexable crossings, and the application of SMCSS, FF is far superior to Ancobar. B and mo alike reduce the pigmentation of the feather. FF-hens are hemizygous to B (B-momo), while cocks are homozygous (BBmomo). This will explain the unique difference in the feather colouring between cocks and hens. B and mo, as it were, come out in full blossom with cocks, turning them almost pure white. However, they always have small gray spots on the neck hackles. G F F cocks have a feature of yellowish red, especially on the shoulders. At the time of moulting, the flowery colouring of FF-hens will be intensified, making the fowls appear still more beautiful in their new plumage. In actual breeding, SFF has turned out to be hardy and, above all, more resistent to Marek's disease. This is due to the fact that SFF has been inbred for more than ten generations and, also, that the breed has not been reared in any real isolation. Strict selection for good phenotype may also have played its part. B. Cock strains Tables 2 and 3 include cock strains of a number of various breeds. However, it is not primarily the different breeds that I want to point out in this respect, but, above all, the many genotypes they represent. White Leghorn, for instance, may easily be varied with regard to genotype by synchronizing different strains, so as to adapt their breeding to SMCSS. SFF has been included in the tables both as cock and hen strain. The cock strain is then taken to be a line that is not included as hen strain, unless a combination of strain breeding and RRS is to be presented in the tabular statements. Norwegian Jaerhons is an old country breed which, as early as 1916, became the object of a beginning systematic breeding. At that time, a breeding centre was started for rearing this breed. It is somewhat smaller than leghorn, and is autosexing from about 1940. For several decades, the breed has been subject to extensive inbreeding. As early as 1915, or thereabouts, there existed highly inbred strains with an annual production of 200 eggs per hen (OLSSON 1930). Jaerhons are still being bred under control in Norway. They are also to be found in Sweden. The breed is characterized by good quality of the eggshell, the colour of the egg often being of a slightly brown shade. A few hens lay rather brown eggs. Crosses between cocks of Jaerhons and hens of Barred Rock are, in Norway, regarded to be just as good layers as White Leghorn ( ESKILT et al. 1958). New Hampshire has existed in Sweden for about 25 years. The best strain in Sweden differs in three respects and in a rather marked way: it is of a smaller and much slender type, it is pure-bred for early feathering, and produces eggs of a darker brown. Some cross-breeds, such as N H X WL and NH X SFF, are still being sold in Sweden. Fiftyfive Flowery in gold (GFF) is a very beautiful breed. G F F cock X S F F hen produces G F F hens of a pure colour. The silver variety, however, attracts considerably more interest. Black Leghorn has never been widely spread in Sweden, but earlier cross-breeds, as Black L. X SFF, used to be very popular. Today Black L. is very rare, and so no crosses of a black type are offered for sale in Sweden. Rhodebar is a barred, autosexing variety of RIR. The breed has been described in Autosexing Annual (LLOYD, HILL and KNIGHT 1949). Black Australorps are found in Sweden in small numbers and have never gained a foothold here. The breed can be used for several crossings of a sex-linked type. Hampbar is a barred variety of NH. A breed of such a light down colour as NH is hard to make definitely sexable only by introducing B into it. However, the breed may well be used for a great number of sexable crossings. Swedish Reddish Brown (SRB) is principally a flowery (mottled, mo) variety of Swedish New Hampshire. The crossbred SRBXSFF is very popular for small stocks, much on account of its white mottled markings. SRB has been produced, in the first place, with increased possibilities with SMCSS in view. Results With a few pedigree strains at disposal it will be possible to make quite a number of RRS-tests, in a short time, by the application of SMCSS. Table 1 comprises 144 combinations for RRS to be performed in less than one year, starting with only four strains (it will, of course, then take some time until the grown-up offspring has been tested and appraised). Tables 1-3 indicate the possibilities of comprehensive and quick RRStests. Each table presupposes the silver variety of Fiftyfive Flowery as hen strain(s). In the four strains, four different cock groups, with three cocks in each, are used. The cock groups, Tables I and 2 (1) All genes concerned have not been included in Table 2. New Hampshire, for instance, is homozygous both for columbian restriction (Co) and dominant wheaten (ewh; SMYTH 1972). Remarks about (2) The particular colour factor characterizing Norwegian Jaerhons has been designated nj. The designation is preliminary. With Jaerhons there also exists a colour type, which, in a typical way, differs from the most common colour type, even in the down colour. (3) In two cock groups cocks are used of the same variety as the hen strain SFF. It is presupposed that the cocks be of a different strain. With cocks of the same strain as the hens, a combination of strain breeding and RRS is obtained. (4) When GFF-cocks are used, all female chicks will turn out purebred GFF-hens, although of a strain crossing type. ( 5 ) All chicks from the 144 combinations, indicated in Table I, are sexable (autosexing and/or sex-linked). (6) As indicated by Table 1 a cock group reappears in the same pen at regular intervals (cock group A, for instance, appears in the same pen in weeks 1, 17, and 33). One of two actions now has to be resorted to, either repeating earlier combinations or exchanging the cocks in respective cock groups for others. As the hens in the different pens are getting too old or, for some reasons, can no longer be used for repeated combinations, replacing the hens at certain intervals will have to be considered. (9) Cocks of White Leghorn may be used with SMCSS, but they will produce a sexable offspring, only with the use of late feathering hen strains (feather sexing). Table 3 indicates the genotype and phenotype of the offspring of the different cocks (of the cocks groups concerned). Also, the type of the sexing method is indicated by a number, explained under "Numerical code for sexing methods". The table presupposes SFF as hen strain, and so do Tables 1 and 3. Table 3 From among autosomal genes only those within E-locus, besides mo an nj, have been included. Remarks about Under "Autosomal genes", nj has been placed as allele to e+, as we have good reasons to assume that nj is an allele to e+. The designation el would, then, be appropriate to the "Jaerhons gene". Concerning "Phenotype of the dayolds", only a very short description has been given. Coloured illustrations will, better than anything, help us to get a clear conception of this type. "Bulletin 38, Sex Linkage in Poultry Breeding" (COLES 1966) includes coloured pictures, which, in many cases, remind you of the colour differences of combinations in Table 3. Under "Sexing method", the number 6, (autosexing and sex-linked) has been assigned to Hampbar X SFF. Hampbar, not being fully autosexing, it is an open question, whether or not it may produce autosexing to 100 %. But the goldsilver difference is so marked, that it will be immaterial whether 100 % autosexing is obtained or not, in a case like this. The male chicks of RRS-combinations are of no consequence, of course, but from a purely genetic point of view it may, nevertheless, be of some interest to have them specified under "Genes that may be accurately identified in the feather". Regarding B and b+ we are actually concerned with BB and Bb+, respectively. The experienced observer will always distinguish between those types in dealing with cross breeds, included in the table. Discussion The tables should be taken to indicate my intention to accentuate different genotypes, rather than The tables should, further, be regarded only as exemplifications of SMCSS. The arrangement of cock groups with three cocks in each does not imply that, in my opinion, this should always be the ideal thing. The philosophy of breeding is today intimately linked toothe "one-cock-system", and the one who introduces SMCSS will probably apply SMCSS without arranging the cocks in cock groups. However, this will not exclude that, by degrees, a fuller and more intricate grasp of the matter will be developed. The length of intervals suggested between shifting cocks (4 weeks) is also preliminary. In one respect or other, intervals of different length may be relevant under varying conditions. Further, the number of strains is, naturally, still more flexible. As a basic rule the flexibility admitted by SMCSS should be utilized in a way that will best suit each particular case. When it comes to breeds, strains and genotypes, numbers will differ in connection with a more comprehensive establishment than with a smaller one. SMCSS should primarily be taken as a system to be used on the basis of an autosexing hen strain with combinations of (1) autosexing and sexlinked type, (2) autosexing type, and (3) sex-linked type. It is very typical for SMCSS, that cocks may be divided into cock groups, that may be shifted continuously without loss of any pedigree of interest, at the same time as there is sexing automation. The concept Multiple pedigree (MPG) is connected with SMCSS, especially if cocks are placed in cock groups as exemplified by the tables. MPG implies that eggs from a pen with two or more cocks may produce a complete pedigree, especially with all female chicks. If, for a pen containing 100 SFF-hens, one cock be supplied of each of the breeds GFF, Jaerhons, BlL, NH, Swedish Reddish Brown, and Rhodebar, each female produced will, through its phenotype, indicate which of the cocks is their father, thus providing for sorting out all male chicks as dayolds. This example goes to show that pens with hundreds of hens may achieve complete pedigree (as far as female chicks are concerned), provided trap-nests are made use of, and the eggs are pedigree hatched. If there are no trap-nests, and consequently the eggs cannot be pedigree hatched, there will still be pedigree on the paternal side, evident in the colouring and the pattern of the female offspring, if not already by the down colour. A variety of MPG may be practised in large pens, namely the use of, say, five full brothers of each breed or variety concerned. Five full brothers of each of six breeds/varieties makes 30 cocks to a pen, which then may have about 400 fowls. When it comes to quickly testing cocks of many different types, MPG is more ideal. In a pen with as many as six cocks (or six groups of full brothers), it will hardly be possible to shift to six others of different genotype and phenotype and, at the same time, retain pedigree accuracy on the paternal side. The example of six cocks is, therefore, not of a SMCSS type. With SMCSS we have, at present, to be confined to using a maximum of 3-4 cocks in each pen. This goes a far way. The examples of the tables with three cocks in each cock group should, as far as the number of the cocks is concerned, prove to be up-to-date for a comparatively long period of time. With more than one cock in each pen, there will also be MPG, with the practice of SMCSS. Hence, MPG is a concept that will always be connected with SMCSS, even if MPG may be used without also applying SMCSS. A breeding institute may very well utilize the possibility of using SMCSS along with MPG, at the same time as MPG is applied to breeding activities apart from SMCSS. Table 3 is a typical example of SMCSS together with MPG. The concept autosexing has not received the popularity it deserves. One reason is that the so-called Cambridge-breeds (including Legbar, Cambar, and Dorbar) have often shown a comparatively low production (HAGEDOORN 1953). But this was due more to White Leghorn having been almost entirely excluded from the autosexing philosophy, and, further, to lack of knowledge and to great deal of misunderstanding about the breeding of autosexing breeds and the production of sexable crosses. HAGEDOORN produced Legbar, autosexing Barnevelder, and Rhodebar through breeding B into Brown Leghorn, Barnevelder and RIR, respectively. Describing the process of production, however, he makes no reference to hen strains having to be in silver, and cock strains in gold, which is one of the most important facts to remember when changing over to autosexing. HAGEDOORN criticizes the method often used in England for the production of new breeds, namely that of taking, for instance, 75 % of the genes from one breed and 25 % from another. He argues that instead of doing so, B gene should be "loaned" to existing breeds so as to create new varieties. But, as a matter of fact, one principle need not exclude the other, because a new breed, with genes from two or more breeds, may, in many respects, be superior to the breed, from which it chiefly stems. It is true, that such goals will not be achieved immediately, but it need not take more than three t o four generations of intensified breeding to prove that the direction chosen is right. HAGEDOORN bred B into Barnevelder, and presents a process chart of the production, which is extended over a period of 14 (!) years, resulting in autosexing Barnevelder with 123/124 of the genes from the Barnevelder strain that was the basis for the production (HAGEDOORN 1953). Charts of this kind are not conductive to creating an interest for autosexing. For a wide application of autosexing, White Leghorn will have to be introduced. There is nothing to indicate that an autosexing form of WL should be inferior in any respect. It would carry us too far to discuss, in this connection, the proper methods for the production of an autosexing WL. This matter will, therefore, be treated separately. It is of interest to note that, due to the fact that cocks, but not hens, may be homozygous for Z-genes (hens are always hemizygous, and so also pure-bred with regard to Z-genes), cocks may be "loaded" more than hens by creating homozygosity for a number of Z-genes in the cocks. This Z-inbreeding may achieve some importance (this may be so, even now), especially if several Z-genes without any negative effect were dis-covered and found to be useful to commercial breeding. Z-inbreeding may be resorted to without application to autosomes, to any great extent. One single outstanding cock, having obtained top results in connection with appraisal of offspring, may be mated with its daughters and produce a strain inheriting its Z-genes from this single cock to 100%. Then the inbreeding may be continued until practically 100 % homozygosity is obtained for the Z-genes within one strain. For a strain to have Z-genes from one cock only does not suffice, since the Z-chromosomes of that cock may, naturally, differ in the setup of genes. There is often homozygosity for genes such as B, S, and Id. With regard to autosexing and SMCSS, homozygosity is necessarily required for certain Z-genes. As more colour genes are discovered, and more data can be laid down about them, the possibilities of utilizing them commercially will be enhanced. In connection with breeding, certain genes are, however, inconceivable on account of their negative effects, in one respect or the other. A few Z-genes are known, the loci of which we do not know as yet. Since genes with loci close to each other are linked to a degree proportional to the distance between the loci, this relationship may be used for practical purposes, as for the production of new breeds/varieties. For the production of Automatic Leghorn, use was made of the close linkage between S and K (SILVERUDD 1968). For obvious reasons, colour genes will, no doubt, be of great importance for all future breeding programmes, including the following aspects: (1) Sexing automation (2) The application of SMCSS and/or MPG (3) As indicators of existing linkages (for instance with blood group genes) (4) As phenotypical characteristics of different strains. In practice this may involve advantages such as facilitating the co-breeding of different strains, without the slightest risk of mixing them. If different strains should be practically identical with regard to phenotype, extreme care must be taken in collecting and storing the eggs as well as hatching and breeding, so as to avoid mixing. ( 5 ) As homozygous setups of genes in pure strains, as opposed to such setups of another colour type in corresponding loci in other strains, thus permanently providing for heterozygosity, when such strains are crossed. Through linkage, homozygosity tends to increase in closely adjacent loci. If a particular gene should lack importance regarding overdominance, an effect of overdominance may be produced through genes that are linked with the gene in question. It is true, that different theories exist of overdominance (see MUNTZING 1971), but in spite of this, there can be no doubt about the importance of heterozygosity in the production of livestock for practical purposes. Quite a number of colour genes would be conceivable for producing new strains that differ in many respects. But there are also many other kinds of genes as, for instance, blood group genes (which may induce overdominance; FALCONER 1960), genes for colour and shape of eggs, quality of shell, size of eggs, type and size of comb, length of down, position of tail, and so on. Colour genes, affecting the colour of ears or eyes, may also be mentioned. Although the value of a planned differentiation of strains can be discussed from various points of view, the basic principles can hardly be rejected. What previously has been said of sexing automation, as well as SMCSS and MPG, represents good examples of the value of differentiation between strains with regard to certain colour genes. Naturally, distinction has to be made between different kinds of differentiation, such as being of advantage for sexing automation, SMCSS and MPG, and promoting higher production, increased hardiness and resistance, higher hatchability, and so on. If two strains are selected on account of highest possible differentiation, and crossed (while retaining the two original strains for comparisons), a better conception may be gained of the advantage of selections of this kind. It is evident that a particular gene may have a quite specific effect. The blood group gene B2 gives a definitely lower mortality than B14 (HANSEN and LAW 1970). SMCSS presupposes that either strain or breed crossing is of current interest. Heterosis in general is so wellknown that, only in passing, it should be emphasized that hatchability, age when starting laying, egg production, and size of eggs will be favourably affected by cross breeding (HUTT 1964). It is also interesting to note, that "the Austra-white hybrids from Australorps X WL are more resistant to respiratory diseases than the parent breeds. This valuable effect of hybrid vigour is also found in crosses between different strains of the same breed" (HUTT 1964). As early as in 1939 Gert Bonnier, Sweden, started experiments that may be considered a beginning to RRS (LILJEDAHL 1973). To-day "Nick-Chick" is nothing less than a trade mark for a hybrid product. Through SMCSS a gene like mo will get a practical significance that it has never had before. And there are several genes that may get a similar significance. Genes like B, S, and s+ have, for very long, proved to be of economic value, although they have not been exploited nearly as much as they deserve. With SMCSS also other genes, besides the colour genes, may come to the fore. A case in point is the gene for rose comb (R), whose presence or absence is evident in dayold chicks, even if it cannot be observed as instantly as many distinct colour differentiations. But genes to be selected should not have any negative effects. Subfertility of cocks homozygous for the auto-soma1 R gene (rose comb) was observed a long time ago. This subfertility of RR cocks is due to an anomaly in the sperms (PETITJEAN 1970). This reduces the interest in R, which, however, is worthy of attention also for other positive reasons. Table 4 presents some examples, indicating how cocks may be shifted when autosexing hen strains are not made use of. The cocks of a particular group may be used simultaneously (without shiftings) or else shifting them one by one (or two by two, in some cases) with full pedigree. In group E there is an Automatic Leghorn (AL) hen strain. AL is to approximately 97 % produced from a couple of old Swedish WL strains. AL cocks are, from a phenotypical point of view, identical with WL cocks, while AL hens have a slight but characteristic tinge on the body, but wholly white neck. AL has neither E nor e+. The colour genes of E-locus cannot be specified and there may be several different genes of yellow/ brown/red type. It is typical for AL that, although Shifting of cocks without loss of pedigree accuracy may be practised also with breeds for meat production, in rare cases when RRS is resorted to. When rearing meat-type breeds, extremely short generation intervals are already used that cannot be shortened appreciably. Through SMCSS it should be possible, to some degree, to accelerate breeding by performing more tests and by the use of more cock strains. A quicker adaptation of the new strains to one another may also be achieved. Z-genes as B, S, s+, Id, and sal, and autosomal, as Db, Co, eWh, c, and I, may be used to colour the strains for production of broilers. Today we know that the genotype ERERCoCoDbDb gives a similar effect as eWheWhCoCo (MOORE and SMYTH 1972).The increased clarification about, for instance, the columbian type (NH and L Sussex are typical representatives) has made it easier to specify genotypes for meat-producing breeds (and, naturally, also for laying breeds and for fancy breeds), which will be of more concern, if there is an eye for autosexing, sex-linked crosses, SMCSS and MPG. "Columbian" genes and genes appearing together with them in some sort of harmony, make out a context, of which we now have more knowledge than previously (cf. SMYTH 1970;SOMES and SMYTH l965,1965b, 1966, 1967SOMES, Fox, and SMYTH 1966;MOORE andSMYTH 1972, 1972b). Since the broiler of today often is of a sex-linked type, the usefulness of SMCSS may very well be considered, even if an ideal colour type may hardly be said to have been presented as yet in any colour combination suitable for broiler production. Autosexing hen strains in silver, and cock strains of, for instance, genotype BB(b+ b+)s+s+CoCoewhewh, are conceivable. When interest is concentrated on 10 to 12 genes, there will be a great variety of more or less conceivable genotypes. I is a problematic gene, if 100 % accurate and rapid sexing is desired. If I is to be included, this should be in cock strains, which often consist of considerably smaller numbers of fowls. With turkeys, ducks and geese there are also Z-linked colour genes (COLES 1966). It will probably be possible to utilize these in a somewhat similar shifting system. Practical advantages Since practical advantages have already been emphasized in this paper, only a summary will now be given, besides a few comments. (1) Continuous cock shiftings without loss of pedigree accuracy may be performed in many different ways. The number of RRS-tests may, in this way, be increased, avoiding having breeding fowls that produce only table eggs, for some time after shifting cocks. (2) More than one cock may be kept in each pen without loss of pedigree. Then "multiple pedigree" (MPG) is applied. (3) Several strains, especially cock strains, may be utilized within the same pen. (4) New strains may, more rapidly, be adapted to each other through an advanced breeding of SMCSS type. Also, the possibilities of starting from a more complicated genotype will be enhanced. ( 5 ) For several loci a constant heterozygosity may be achieved by crossing the strains that are homozygous for different alleles in homologous loci. (6) SMCSS will reduce the cost per test of RRS-type or similar tests, also when applied in part. Since SMCSS is closely connected with sexing automation, emphasis should also be laid on the advantages of high class sexing. (7) Lower sexing costs (or, at best, none) both regarding pure autosexing strains and crosses with them. (8) Special sorting personnel will not be required, nor sorting contracts etc. (9) Diverse paraphernalia may be dispensed with, such as tables, lamps, apparatuses etc. (10) Risk of infections through sexing operators is completely eliminated (higher mortality through coli bacteria, for instance, has been observed in some cases). (1 1) The collection and sexing of dayold chicks will be reduced to one action. (12) With pedigree hatching the registration of males and females separately, in connection with marking, will be facilitated. It will be easier to escape marking those males that are not to be used for breeding, or only for the production of fowls for sale. (13) When dayold female chicks are sold for breeding purpose, there will be no risk of a male slipping through, to be used by the buyer for further breeding. (14) Without being hampered by the need of sexing operators, chicks may be picked out from an incubator at a somewhat earlier stage, for instance, when a consignment is to be dispatched over a long distance and, therefore, has to be sent as early as possible. The flexibility of SMCSS should be pointed out. Thousands of varieties are conceivable. For one thing, this will be important, with regard to the results that future research may arrive at. The usefulness of SMCSS is linked up with that of RRS. In a special way, it provides for "continuous breeding", so as to facilitate continuous shiftings without loss of pedigree accuracy in connection with any planned development. The importance of "continuous heterozygosity" deserves special attention. Research on this subject may lead to discovery of some facts of special interest and importance for poultry breeding. The great interest existing for blood group genes should also include colour genes far more than hitherto. In the past, colour genes have received attention as indicators of linkage. If, for instance, a linkage is found between a colour gene and a desirable blood group gene, this will involve, for instance, that, in producing a new strain to be a carrier of the blood-group gene concerned, a number of fowls of the desired genotype may be obtained quicker, or, at least, with a smaller number of tests. When such a blood-group gene appears in a low frequency, in producing a new strain, the linkage with a colour gene may be of great importance. The importance of sexing automation for the hatcheries has not received due attention. In the combination of autosexing and sex-linked sexing, a group of, say, 1000 chicks may be picked out from an incubator, sexed, counted and packed into boxes in about 15 minutes. The male chicks will, of course, not be counted. The fact that we are living in the computer age will make it possible for us to utilize the theoretical possibilities with SMCSS in a way that would not otherwise be possible. The computer adds to the importance of SMCSS, and SMCSS adds to the importance of the computer.

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2020-12-10T09:04:12.335Z

1974-04-01T00:00:00.000Z

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Distribution of Zearalenone-Producing Fusarium Species in Japan One hundred sixty-six isolates of Fusarium spp. from domestic cereal grains, feed, and other sources were examined for their ability to produce zearalenone on autoclaved moist rice grains. They belonged to the following species (number of producers/number tested): F. roseum (9/28), F. roseum (Culmorum) (3/4), F. roseum (Gibbosum) (2/5), F. roseum (Avenaceum) (1/2), F. roseum (Scirpi) (0/1), F. tricinctum (1/4), F. tricinctum (Sporotrichiella) (0/7), F. lateritium (1/1), F. episphaeria (0/2), F. moniliforme (0/3), F. oxysporum (0/12), F. rigidiusculum (0/4), F. solani (0/4), F. splendens (0/1), F. nivale (0/2), and Fusarium spp. (15/86). Zearalenone was isolated from molded rice by ethanol extraction and purified by column chromatography. Selected isolates of F. roseum M-3-2 and F. roseum (Gibbosum) A-O-2 produced 50 to 100 mg of zearalenone per kg of rice. Increased yields (250 to 407 mg/kg of rice) were obtained by F. roseum M-3-2 when the substrate was supplemented with 1% peptone. In previous experiments, toxicological and microbial surveys on trichothec mycotoxins of Fusarium spp. were performed in connection with "Akakabi (scabby grain) toxicoses" of men as well as "bean-hull poisonings" of horses and farm animals (14)(15)(16). Consequently, nivalenol (11), fusarenon-X (18,19), T-2 toxin, and neosolaniol (5,15), all of them having 12-13 epoxy-trichothecene nucleus and potent inhibitors of protein synthesis in animal cells, were detected among the metabolites of F. nivale, F. solani, F. sporotrichioides, and others (17). During the course of this survey, we attempted to clarify the distribution in Japan of fusarial strains that produce zearalenone, a uterotrophic mycotoxin of resorcylic acid lactone (20). We report here an assessment of the wide distribution of the zearalenone producer, environmental factors influencing mycotixon production, and a preparative procedure for isolation of the mycotoxin from moldy rice grains. (This investigation was presented in part at the 24th Annual Meeting of the Japanese Food Hygienic Society, Akita, Japan, 12 wheat, rice grains, rice straw for bedding in stables, river water, and other sources from the districts where fusarial toxicosis of men and farm animals were sporadically reported (13). Several strains were kindly supplied by H. Kurata (National Institute of Hygienic Sciences), N. Morooka (Kagawa University), and Y. Matsuda (Public Health Research Institute of Koube City). Identification of the species was carried out by one of the authors (H. Tsunoda). according to the taxonomic system of Toussoun and Nelson (12) and that of Bilai (1). All the strains were subcultured on potato-dextrose agar for 14 days at 25 C, followed by storage at 4 C. Detection of mycotoxin. Zearalenone was extracted and assayed by the method of Eppley (4) with the following modifications. The laboratory-molded rice powder (50 g) was extracted for 30 min with a mixture of 25 ml of water and 250 ml of chloroform in a high-speed blender, and upon filtration the first 50 ml of chloroform extract was charged on a column (2.2 by 30 cm) that consisted of three layers of 5 g of Na2SO4, 10 g of silica gel, and again 15 g of Na2SO4. The column was eluted with 150 ml of n-hexane, 150 ml of benzene, and 250 ml of benzene-acetone (95:5), the last eluate was evaporated to dryness, and the residue was dissolved in 1 ml of benzene. Silica gel (kiesel gel G, Merck) thin-layer plates (0.25 mm, activated for 30 min at 110 C) were spotted with 20 ,gliters of the benzene solution. The plates were developed with the following solvent systems: benzene-acetic acid (9: 1), benzene-acetone (9: 1), or chloroformethanol (95:5), and inspected under an ultraviolet lamp at 254 nm. Quantitative analysis. For quantitative analysis, zearalenone was extracted by the Eppley procedure (4) and separated by thin-layer chromatography (Kiesel gel HR, Merck; 0.5 mm in thickness). After development with the solvent n-hexane-acetone (4:1), zearalenone-containing gel was scrached from the plates and extracted with methanol, and the content was estimated from the absorption at 236 and 274 nm with a molecular absorption coefficient of 29,700 (236 nm) or 13,900 (274 nm) (20), or an optical density of 0.46 (236 nm) for 5 gg of zearatenon solution per ml. Instrumental analysis. Melting points were determined with a Mitamura micro-melting-point determinator. Infrared spectra were taken on KBr pellets with a Hitachi model 225 infrared spectrophotometer, and ultraviolet spectra were measured in methanol with a Hitachi model 323 recording spectrophotometer. RESULTS AND DISCUSSION Screening for zearalenone producers. Initially, 166 isolates of Fusarium were screened for the ability to produce zearalenone when grown on rice grains ( Table 1). The mycotoxin was detected from 32 isolates such as F. roseum, F. roseum (Culmorum), F. roseum (Gibbosum), F. roseum (Avenaceum), F. lateritium, and F. tricinctum. These results were the same, except for F. lateritium, as those reported by R. W. Caldwell et al. (2). Zearalenone producers were distributed in several districts of Japan and were isolated from oats, barley, wheat, bean hull, and river water ( Table 2). Isolates from rice and rice paddies did not produce zearalenone, although rice is an appropriate substrate for zearalenone production. These findings strongly indicated that the zearalenone-producing fungi are distributed in a wide range of cereal grain as well as feed. This is the first report on the distribution of zearalenone-producing fungi in Japan. In the United States, this mycotoxin has been found in the feed of dairy cattle and is suspected of playing some role in the infertility problems of these animals (8). However, a correlation between the consumption by animals of zearalenone-contaminated scabby grain and abortion and infertility remains to be established (9, 10). Isolation procedure of zearalenone from the moldy rice. Purification and isolation procedures of zearalenone are schematically represented in Fig. 1. F. roseum M-3-2 was inoculated on partly polished rice grains, and, after cultivation for 14 days at 25 C followed by 14 days at 12 to 15 C, the moldy rice was dried at 60 C overnight and powdered. The powder (3 kg) was continuously extracted with n-hexane for 10 to 14 h with a Soxhlet-type extractor, and the defatted residue was extracted twice with 5 liters of ethanol. The combined reddish-yellow ethanol extract was evaporated to a syrup. The residue was suspended in 1 liter of water and then extracted with two 500-ml portions of benzene. Upon evaporation of the solvent, 3.2 g of the oily material was obtained. The first purification of the crude extract on a Kiesel gel column (4 by 45 cm) with 1.5 liter of benzeneacetone (9:1) and the second chromatography with 1 liter of chloroform-methanol (99:1) resulted a yellow powder. Upon crystallization with chloroform and n-hexane, white needles Were deposited with a yield of 30 to 40 mg/kg of rice (melting point 161 to 162 C; maximal absorbancy 236, 274, and 314 nm). These data and the infrared spectrum (6) confirmed the needles as zearalenone. Under the same conditions and procedures, F. roseum Map. 10 (the strain from C. J. Mirocha) produced 50 to 130 mg of zearalenone/kg of rice. Effect of ingredients on zearalenone pro-duction on rice. The effect of supplements on zearalenone synthesis was examined with rice grains as substrate. F. roseum M-3-2 was inoculated on rice supplemented with 1% (wt/wt) of various nutrients; after cultivation for 14 days at 25 C followed by 14 days at 15 C, the zearalenone concentration was spectrophotometrically determined at 236 and 274 nm after separation by thin-layer chromatography. The addition of peptone or yeast extract increased the mycotoxin concentration six-and twofold, respectively, over the control (Table 3). Glucose, malt extract, or meat extract was found to suppress the synthesis. Since peptone addition did not result in increased growth, it is presumed that it affected enzyme synthesis required for toxin production. We have no basis for this supposition, for it is conceivable that enzyme derepression could also have occurred. An additional 14 days of incubation at 25 C after the 12 to 15 C period (7) resulted in increased zearalenone production in both unsupplemented and peptone-supplemented rice (Table 4). 'Average of duplicate. Production of zearalenone on liquid media. Zearalenone production was evaluated on two liquid media. F. roseum (Gibbosum) A-0-2 was inoculated in 500-ml Fernbach flasks, each containing 300 ml of the culture substrate. After cultivation in standing culture, the culture filtrate was extracted with benzene, and the dried mycelia were first defatted by n-hexane followed by extraction with benzene and sequently ethanol. A very small amount of zearalenone was detected by thin-layer chromatography, only in the benzene-soluble extract of mycelia. This lower yield of mycotoxin suggests that the nutritive value of the solid is more conducive to toxin synthesis.

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2020-12-10T09:04:12.285Z

1974-03-01T00:00:00.000Z

237234982

s2

Interaction of pH and NaCl on Enumeration of Heat-Stressed Staphylococcus aureus The effect of pH level and NaCl concentrations, alone and in combination, on the enumeration of unstressed and heat-stressed cells of three strains of Staphylococcus aureus was determined. A definite narrowing of the optimum pH range for enumeration of both unstressed and heat-stressed cells was observed as the NaCl concentration was increased from 0.0 to 7.5%. Counts of unstressed cells diminished only slightly with increases in NaCl, whereas heat-stressed cells showed a marked sensitivity to NaCl concentrations of 4% and above, regardless of the pH level. Because of this sensitivity to NaCl, recoveries were far poorer than with unstressed cells at NaCl concentrations of 4% and above. The ability of Staphylococcus aureus to grow at greater NaCl concentrations in comparison to many other bacteria is well documented (10,12). A number of media that use NaCl as the basis of their selectivity have been developed for the quantitative enumeration of this organism (3,4). In some situations, enumeration of S. aureus by such selective media may be difficult if the organism has been subjected to heat treatment or other forms of physical stress sufficient to cause sublethal injury. However, the assumption is often made that conditions satisfactory for the enumeration of unstressed bacteria are equally applicable to heatdamaged bacteria. Busta and Jezeski (2) found that S. aureus cells heated at 60 C for various lengths of time lost their ability to multiply on Staphylococcus medium 110 containing the normal concentration of 7.5% NaCl. Since then other investigators (8,13,15,17) have reported similar observations, indicating a definite NaCl sensitivity by S. aureus after thermal injury. Such a change in NaCl tolerance obviously reduces the effectiveness of media that are designed to selectively enumerate S. aureus on the assumption that the organism is tolerant to high concentrations of NaCl. In addition to a loss of NaCl tolerance after thermal injury, F necessary for the recovery of heat-stressed S. aureus was reduced in comparison to that for the unstressed organism. Other reports on the effects of pH alone or pH in combination with NaCl have been concerned with unstressed S. aureus. For example, Lechowich et al. (11) found that pH 5.6 reduced both aerobic and anaerobic growth of S. aureus in pickling brines used for curing meats, and pH 4.8 prevented growth. landolo et al. (9) demonstrated that the effects of NaCl on unstressed cells were greater either when pH deviated from the optimum or incubation temperature was raised to 45 C. Genigeorgis and Sadler (7) concluded that growth and enterotoxin production were better when initial pH of the medium was increased and salt concentration was decreased. Growth of organisms tended to raise the pH so that the pH at time of enterotoxin production unquestionably was higher than when growth was initiated. Scheusner et al. (14) obtained growth when the initial pH of the broth was 4.96 to 9.02. Genigeorgis et al. (5) reported growth over the pH range from 4.00 to 9.83 in the absence of NaCl, but increasing concentrations of NaCl narrowed the range of both acid and alkaline values, the limits being pH values of 4.50 and 8.0 at 12% NaCl. Nelson (Bacteriol. Proc., p. 20, 1971) reported that the inhibitory effects of pH and NaCl were additive when enumerating heat-stressed coliform bacteria, maximum counts being obtained only over a pH range of 6.0 to 7.0 in the presence of 3% NaCl, whereas unstressed organisms were unaffected over a pH range from 5.0 through 9.2 in the absence of NaCL in the enumeration medium. MATERIALS AND METHODS Test organisms. Three strains of coagulase-positive S. aureus were employed. Strain UA-112 was obtained from the Department of Microbiology and Medical Technology at the University of Arizona. Strains S-6(B) and FRI-100, known producers of enterotoxin, were obtained from M. S. Bergdoll of the Food Research Institute of the University of Wisconsin. The organisms were maintained on brain heart infusion agar (Difco) slants held at 5 C prior to use. Cells were grown in 5 ml of nutrient broth (Difco) for 24 h at 37 C, after which 1 loopful was transferred to 50 ml of nutrient broth contained in 125-ml flasks and incubated for 24 h at 37 C. Thermal stressing and enumeration. A 1-ml sample of a 24-h culture was placed into 5 ml of sterile reconstituted milk (110 g of milk solids-not-fat/liter) contained in screw-cap test tubes (16 by 125 mm). A sample serially diluted in phosphate buffer was plated on unmodified nutrient agar (Difco) by the procedure outlined in Standard Methods (1). Cells of UA-112 were plated on nutrient agar containing NaCl concentrations of 0, 2, 3, 3.5, 4, 5, and 7.5%. Counts at each NaCl concentration were made at pH levels of 5.0, 5.5, 6.0, 7.0, 8.0, 8.5, 9.0, and 10.0. Cells of strains S-6(B) and FRI-100 were plated on nutrient agar containing fewer NaCl concentrations accompanied by fewer pH levels to check only those points of greatest importance. NaCl concentrations of 0, 2, 3, 3.5, 4, and 5% were used, along with pH levels of 5.0, 5.5, 7.0, 7.5, and 9.0. Adjustment of pH was made prior to plating by the addition of either 1 N H2S04 or 1 N KOH to the warm NaCl-nutrient agar. After the plating of the unstressed cells, the remaining milk sample was immersed, along with a control milk sample containing a thermometer, in a water bath maintained at 56 ± 0.1 C. Timing of the sample commenced when the temperature reached 56 C in the control tube. Exposure time of the sample was designed so as to reduce the viable population as determined on nutrient agar by 99% and was 7 min for strain UA-112 and 6 min for strains S-6(B) and FRI-100. A high level of kill was selected to maximize the observed effects of stress. After exposure, samples were cooled in ice water and plated on the same series of media on which the unstressed cells had been plated. Plates were incubated at 37 C for 48 h. All platings of strain UA-112 were made in duplicate, and two runs were made for each NaCl-pH combination. Duplicate platings for strains S-6(B) and FRI-100 were used only for counts on the control unmodified nutrient agar. Two trials were run for each NaCl-pH combination. Statistics. Counts of stressed cells on modified media were expressed as a percentage of the count obtained on unmodified nutrient medium, and this percentage then converted to logarithms to the base 10 to give log percent recovery. Recovery values for unheated and heated S. aureus UA-112 were subjected to a completely randomized two-way factorial arrangement (16) by using NaCl and pH as the factors. A similar experimental analysis for unheated and heated cells of strains S-6(B) and FRI-100 involved a completely randomized three-way factorial analysis of variance, with NaCl, pH, and strain constituting the factors. RESULTS Data on recovery of unheated cells of S. aureus strain UA-1 12 at different levels of pH in the presence of increasing concentrations of NaCl are shown in Fig. 1. A gradual narrowing of the optimal pH was shown as the NaCl concentration increased from 0 to 7.5%. At lower NaCl concentrations, maximum recoveries were obtained over a pH range of 5.0 through 9.0. At NaCl concentrations of 3.5 and 4%, recoveries at pH 9.0 were significantly less than maximum, whereas recoveries at 5 and 7.5% were significantly reduced at pH levels of 5.0, 5.5, and 6.0. Maximum recoveries at higher NaCl concentrations were only slightly lower than recoveries at the same pH levels in the absence of NaCl. Data on recovery of heat-stressed S. aureus strain UA-112 at different pH levels in the presence of increasing concentrations of NaCl are shown in Fig. 2. These heat-stressed cells also showed a gradual narrowing of their optimal pH range as the NaCl concentration increased. Recoveries at 4% NaCl showed an overall decrease from counts obtained at 3.5% regardless of the pH level. Recoveries at 5 and 7.5% NaCl were quite similar to those obtained at 4% in that recoveries were only approximately 10% of the optimum. Recoveries of unstressed and heat-stressed cells of strains S-6(B) and FRI-100 at various pH levels in the presence of increasing NaCl concentrations were so similar to those of strain UA-112 that the data are not presented. Analysis of variance for all three unstressed strains of S. aureus revealed significant firstorder interactions between NaCl and pH, indicating that changes in the level of one factor modify the effects of the other factor on the enumeration of unstressed cells. In addition, comparison of unstressed cells of strains S-6(B) and FRI-100 showed no significant differences between these two strains in their responses to NaCl and pH. Heat-stressed cells of all three strains also showed significant first-order interactions between NaCl and pH, indicating an interactive effect of these two factors on enumeration. Furthermore, comparison of strains S-6(B) and FRI-100 to one another revealed two additional first-order interactions. Significant interactions were noted with the pH and strain and also NaCl and strain factors. These interactions indicate that the two strains reacted slightly differently to pH and over all NaCl concentrations and to NaCi over all pH levels. In comparison to unstressed cells, heat-stressed cells showed more variation from strain to strain in their responses to NaCl and pH, even though both groups show a narrowing of their optimum pH range as the NaCl concentration increases. DISCUSSION Although three strains do not constitute a large sample, the indications are that the responses to pH and NaCl demonstrated here are characteristic of the species. Such responses to pH and NaCl indicate an interaction effect by the two factors on recovery of S. aureus. As the degree of adversity, especially pH, was increased, a gradual decline in count occurred; this decline was accentuated by increasing NaCl concentration. Both unstressed and heatstressed cells showed a narrowing of their optimum pH ranges as the NaCl concentration was increased. The degree of the observed effects very probably could be altered appreciably by variations in the degree of stress. These findings were in close agreement with the results obtained by Genigeorgis et al. (5,6) on unstressed cells of S. aureus, indicating a decrease in count of the organism at any pH level as the NaCl concentration increases. However, due to the marked sensitivity of heat-stressed cells to NaCl, overall recoveries at 4% NaCl and above were far poorer, regardless of pH, in comparison to unstressed cells. Maximum counts of unstressed cells of S. aureus can be obtained quite effectively in a medium containing 7.5% NaCl adjusted to a pH range of 7.0 through 8.5. Maximum counts of heat-stressed cells cannot be attained on media containing 5% or more NaCl. The pH of the medium plus sample should not be outside a range of 5.5 through 8.5, even with an NaCl concentration below 4%. Although dropping the NaCl concentration of a selective medium would obtain higher counts of staphylococci that had been subjected to thermal stress, it would do away with much of the selective character of such a medium. Due to the NaCl sensitivity of S. aureus after thermal stress, it would appear that no combination of pH and NaCl would offer promise of improving recovery of such an organism from mixed populations of bacteria. Improvements of media for this purpose must be sought in other directions.

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2020-12-10T09:04:12.369Z

1974-05-01T00:00:00.000Z

237229656

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Acid Protease Production by Fungi Used in Soybean Food Fermentation Growth conditions for maximum protease production by Rhizopus oligosporus, Mucor dispersus, and Actinomucor elegans, used in Oriental food fermentations, were investigated. Enzyme yields by all three fungi were higher in solid substrate fermentations than in submerged culture. The level of moisture in solid substrate must be at about 50 to 60%. Very little growth of these fungi was noted when the moisture of substrate was below 35%, whereas many fungi including most storage fungi generally grow well on solid substrate with that level of moisture. Among the three substrates tested—wheat bran, wheat, and soybeans—wheat bran was the most satisfactory one for enzyme production. The optimal conditions for maximum enzyme production of the three fungi grown on wheat bran were: R. oligosporus, 50% moisture at 25 C for 3 to 4 days; M. dispersus, 50 to 63% moisture at 25 C for 3 to 4 days; A. elegans, 50 to 63% moisture at 20 C for 3 days. Because these fungi are fast growing and require high moisture for growth and for enzyme synthesis, the danger of contamination by toxin-producing fungi would be minimal. Growth conditions for maximum protease production by Rhizopus oligosporus, Mucor dispersus, and Actinomucor elegans, used in Oriental food fermentations, were investigated. Enzyme yields by all three fungi were higher in solid substrate fermentations than in submerged culture. The level of moisture in solid substrate must be at about 50 to 60%. Very little growth of these fungi was noted when the moisture of substrate was below 35%, whereas many fungi including most storage fungi generally grow well on solid substrate with that level of moisture. Among the three substrates tested-wheat bran, wheat, and soybeans-wheat bran was the most satisfactory one for enzyme production. The optimal conditions for maximum enzyme production of the three fungi grown on wheat bran were: R. oligosporus, 50% moisture at 25 C for 3 to 4 days; M. dispersus, 50 to 63% moisture at 25 C for 3 to 4 days; A. elegans, 50 to 63% moisture at 20 C for 3 days. Because these fungi are fast growing and require high moisture for growth and for enzyme synthesis, the danger of contamination by toxin-producing fungi would be minimal. Aspergillus, Rhizopus, Mucor, and Actinomucor have long been used in Oriental food fermentations (3). Extensive studies have been made on soy sauce and miso fermentations carried out by Aspergilli, but not on those food processes involving Rhizopus, Mucor, and Actinomucor. During the last decade, pure culture fermentation methods for making tempeh from soybeans by Rhizopus oligosporus (2,8) and sufu from soybeans by Mucor dispersus and Actinomucor elegans (12) were developed. We have also expanded our effort to explore the enzymes produced by these fungi, partly to find out more about the role of such enzymes in these fermentation processes and partly to discover commercially useful enzymes. Our previous studies revealed that R. oligosporus (14), M. dispersus (13), and A. elegans (unpublished data) all produce acid type proteases, whereas most fungal proteases have a pH optimum around neutral or alkaline. Although these fungi grew abundantly in submerged culture containing soybeans, wheat, or wheat bran, their enzyme yields were unsatisfactory. The production of proteases by R. oligosporus decreased as the concentration of substrate increased (16). The protease produced by M. dispersus NRRL 3103 (formerly M. hiemalis) was bound to the mycelial surface (13). Addition of sodium chloride or other 901 ionizable salts in the growth medium for this fungus increased the enzyme in the culture filtrate. The total enzyme yield, however, was limited because fungal growth was suppressed by the added salt. The low enzyme production by these fungi in submerged culture was not unexpected, because fermentation processes carried out by these fungi are usually in solid state. Before World War II, solid state fermentation, generally known as the "bran process," was almost universally employed for the production of fungal enzymes. Deep-tank fermentation, however, has since replaced this technique in the West. Success in obtaining high yields of secondary metabolites by some fungi on solid substrates (4) and difficulties encountered in increasing enzyme yields of the fungi under investigation in submerged culture prompted us to return to our work with solid substrates. The present study was undertaken to find a set of conditions for protease production by R. oligosporus, M. dispersus, and A. elegans on solid substrate. MATERIALS AND METHODS Cultures. R. oligosporus NRRL 2710, M. dispersus NRRL 3103, and A. elegans NRRL 3104 were maintained on slants of potato-dextrose agar at 4 C. Before each experiment, the organisms were transferred to slants that then were incubated at 25 C for 7 days. Spore suspensions for inoculation were prepared by adding 3 ml of sterilized distilled water to each slant and vigorously shaking the culture for 1 min. Fermentation. Each 300-ml Erlenmeyer flask containing 10 g of wheat bran and 4, 8, or 15 ml of water was mixed and allowed to stand for 1 h at room temperature with frequent shaking. The flasks were autoclaved at 120 C for 20 min, cooled, and inoculated with 0.2 ml of inoculum. The cotton plugs were covered with aluminum foil to prevent evaporation, and flasks were incubated stationary at 15, 20, 25, or 32 C for varying lengths of time. The fermentation mass was extracted with 100 ml of 2% sodium chloride solution at room temperature for 1 h with frequent stirring, followed by centrifugation. The supernatant was used as the source of protease. Two experiments were carried out for each set of conditions. The average values of two runs will be presented. Assay of proteolytic activity. Proteolytic activity was measured according to the hemoglobin digestion method described by Anson (1). Reaction mixture containing 1 ml of 1% denatured hemoglobin in 0.05 M citrate buffer of pH 2.5 and 1 ml of properly diluted culture extract was incubated at 38 C for 20 min. The reaction was stopped by the addition of 3 ml of 5% trichloroacetic acid. The undigested hemoglobin was removed by filtration, and the acid-soluble products were determined spectrophotometrically at 280 nm. One unit of protease is defined as the amount of enzyme that yields a change in optical density at 280 nm equivalent to 1 Mmol of tyrosine per h at 38 C. RESULTS Moisture content of wheat bran medium. The initial moisture content of autoclaved wheat bran media containing 10 g of bran and 4, 8, or 15 ml of water was determined by drying at 110 C for 24 h. The average values obtained from three experiments were 35, 50, and 63%, respectively. Visual growth as affected by incubation temperature and moisture of wheat bran. Quantitative determination of growth on solid substrates, such as wheat bran, is difficult. Therefore, only subjective observations on growth are presented in Table 1. Of the three wheat bran media studied, the medium having the lowest moisture, 35%, did not support good growth of R. oligosporus or A. elegans at any of the temperatures investigated, although growth usually was noticeable after 2 weeks of incubation. M. dispersus grew fairly well on wheat bran of 35% moisture at 25 and 32 C, but it grew slower at 20 C. On the other hand, wheat bran media containing 50 and 63% moisture provided all three fungi with an environment for luxuriant growth. Rates of growth, 27,1974 however, were affected by the incubation temperatures and also varied between the three fungi. R. oligosporus grew rapidly on moist media of 50 and 63% moisture. Abundant growth was observed after 1 day at 32 C, 2 days at 25 C, and 4 days at 20 C. M. dispersus did not grow as rapidly as R. oligosporus at the highest temperature, 32 C, but it surpassed the growth of R. oligosporus at the lower temperature of 20 C. A. elegans also grew well at low temperature, and it preferred to grow on the medium with 63% moisture. Production of acid protease by R. oligosporous NRRL 2710. The amounts of acid protease produced by R. oligosporus grown on wheat bran containing 50 and 63% moisture and at three different temperatures are summarized in Fig. 1. The data on enzyme production by the fungus on 35% moisture bran are not presented, because the activities were low throughout the 2 weeks of incubation. As indicated in Fig. 1, enzyme activities reached a maximum after 2 to 3 days at 32 C, 3 to 4 days at 25 C, and 5 to 7 days at 20 C. After that, the activities decreased. The rate of inactivation, however, seemed to be slower at 20 C than at the other two temperatures. A large shift in culture pH values was noted. An initial pH value of 5.7 usually rose to above 7 as enzyme production reached its maximum and continued to rise gradually. Although the fungus appeared to grow as well on wheat bran containing 63% moisture as on that of 50%, the amount of enzyme recovered from media of 50% moisture was much greater than that from media of 63% moisture. Under the conditions investigated, R. oligosporus grown on wheat bran of 50% moisture for 3 to 4 days at 25 C yielded the highest amount of protease. Our results also indicated that the moisture content of the medium was a more important factor than incubation temperature. Production of acid protease by M. dispersus NRRL 3103. As stated before, M. dispersus grew fairly well on bran containing 35% moisture; the amount of enzyme produced, however, was insignificant during the 2 weeks of incubation. Thus, only the results obtained from the growth media containing 50 and 63% moisture are presented in Fig. 2. When M. dispersus was grown on either 50 or 63% moisture bran, it produced equally impressive amounts of enzyme at growth temperatures of 20 or 25 C, but significantly less at 32 C. Production of enzyme by the fungus grown on 50% moisture bran reached a steady maximum after 4 to 6 days at 25 C and 7 to 9 days at 20 C, and then gradually decreased. At 63% moisture, the protease yield was maximum at about 3 days at 25 C and 7 to 8 days at 20 C, and then rapidly decreased to about zero. It is apparent that M. dispersus can tolerate a broad moisture range for enzyme synthesis. The enzyme, however was increasingly susceptible to denaturation as the percentage of moisture increased. Therefore, excellent yields of enzyme by M. dispersus can be recovered from bran containing 50 to 63% moisture after 3 to 4 days of growth at 25 C, or for longer incubation time at lower temperatures. The pH of extract from fermented wheat bran at 63% moisture was above 7 after 2 weeks of incubation; whereas at 50% moisture, it was around 6 except when the culture was incubated at 32 C. Here the pH also reached 7 after 2 weeks. Production of acid protease by A. elegans NRRL 3104. Of the three fungi studied, A. elegans required the lowest temperature for growth and enzyme production. Like R. oligosporus and M. dispersus, this fungus did not grow well on 35% moisture bran nor did it produce meaningful amounts of enzyme. However, when the moisture of bran was high enough for good growth, the organism showed a marked degree of moisture tolerance for enzyme production as indicated in Fig. 3. The enzyme yield, on the other hand, was greatly affected by temperature. The fungus grew luxuriantly at 25 C yet produced low enzyme yield. In this respect, A. elegans behaved similarly to M. dispersus. Unlike M. dispersus, a marked pH shift from 5.6 to 7.9 of A. elegans culture extracts was observed under all the conditions investigated, and the enzyme decreased rapidly regardless of the moisture content of the media. Based on this study, the optimal conditions for protease production by A. elegans grown on wheat bran were 50 to 63% moisture with 3 days of incubation at 20 C or 4 to 5 days of incubation at 15 C. Effect of substrate and culture method on protease production. Like wheat bran, soybeans or wheat did not provide a good growth condition for R. oligosporus, M. dispersus, and A. elegans unless the moisture level of these materials was around 50%. The protease yield per unit substrate produced by these fungi grown on solid state and in submerged culture is presented in Table 2. Wheat bran generally was a superior substrate for enzyme production by these fungi regardless of the culture method employed. Solid culture fermentation was a better method than the submerged culture fermentation. DISCUSSION Many members of the order Mucorales are known to be hydrophytes. They require a relative humidity of 90% or greater for growth and grow best at humidities near 100%. However, little is known regarding the relationship between their growth conditions and enzyme yields. The three fungi investigated in this study, representing three genera in Mucorales, were found to be hydrophytes and fast-growing. R. oligosporus is used for tempeh fermentation. M. dispersus and A. elegans are used for sufu fermentation. The substrates for these fermentation processes have moisture contents of about 55 and 80%, respectively. Therefore, the high moisture requirement by those fungi for growth is expected. The relationship between enzyme yield and the three environmental factors investigated varied with the organism, but they all yielded greater amounts of enzyme at temperatures lower than their optimum growth temperatures. Similar findings with respect to protease production by other fungi were reported by Max- Submerged culture: 10 g of substrate, soybean meal, wheat flour, and wheat bran in 100 ml of water for R. oligosporus, and in 100 ml of 0.5 M NaCl for M. dispersus and A. elegans. Incubated on a reciprocating shaker at temperature and time same as for solid culture. well (6) using Aspergillus oryzae, and Yamamoto (17) using Aspergillus sojae. The effect of temperature on enzyme production observed in this study also emphasized the importance of some common practices in solid culture fermentation, i.e., frequent turning of the growth mass or use of thin layers of solid substrates. Otherwise, the heat resulting from active growth will increase the incubation temperature and affect the enzyme production. As with most extracellular enzymes produced by many microorganisms, maximum yield by the three fungi studied was usually reached about the time of maximum growth. However, the enzymes were rapidly inactivated except when M. dispersus was grown on wheat bran containing 50% moisture at 20 and 25 C. Although many enzymes are often destroyed by protease produced in the same culture, the disappearance of protease observed in this study probably was not due to self-digestion. Previously, we have reported (15) that no degraded products of self-digestion acid protease isolated from culture filtrate of R. oligosporus could be detected when the enzyme preparation was incubated at 28 C for 25 h. The inactivation of the protease was explained as being caused by an alkaline shift in pH. Acid proteases produced by R. oligosporus and M. dispersus are very unstable as the pH approaches 7 (13,14). The rapid disappearance of the proteases limited the harvest time. On the other hand, this trait can benefit the yield and purification processes for other useful enzymes produced by the same organisms. There are conflicting reports on the importance of aeration for the production of enzymes by microorganisms. Richou and Kourilsky (7) found that, in general, aeration was unfavorable for protease formation by various microorganisms. Our study also suggested that aeration may not be an important factor for enzyme production by the three fungi investigated. We noted that the very moist substrate and dense mycelial growth resulted in a tight mass that restricted aeration. The finding that a solid substrate method for protease production by these fungi was superior to the commonly used liquid culture method was not unexpected, mainly because these fungi have been traditionally used in.solid substrate fermentation. There have been few comparisons by these two methods of enzyme yield per unit substrate. Recently, Tsujisaka et al. (11) found that Aspergillus niger produced more lipase on solid medium containing wheat bran and calcium carbonate than in liquid media. It is, however, surprising to find that soybeans were not as good a substrate for protease production as was wheat bran. The fact that these fungi are fast growing and require high moisture levels to grow and synthesize enzymes might eliminate the danger of contamination by such toxin-producing fungi as Aspergillus flavus and Aspergillus ochraceus, which have been reported (5,9,10) to require low moisture levels (calculated at 33%) for toxin synthesis. This should improve the feasibility of using Mucor crude enzyme preparation in food and feed industries.

v3-fos

2019-03-19T13:13:20.058Z

1974-11-01T00:00:00.000Z

237231486

s2

Rapid Methods for Biochemical Testing of Anaerobic Bacteria Rapid biochemical tests for nitrate, indole, gelatin, starch, esculin, and o-nitrophenyl-β-D-galactopyranoside were performed on 112 strains of anaerobic bacteria. All tests were incubated under aerobic conditions, and results were recorded within 4 h. The tests for nitrate, indole, and starch showed a 95% or greater correlation when compared to the standard biochemical tests. Tests for esculin and gelatin showed an agreement of 86 and 77%, respectively. PathoTec test strips for nitrate, indole, esculin, o-nitrophenyl-β-D-galactopyranoside, Voges-Proskauer, and urease were also tested and showed encouraging results. The development of simple, rapid, and sensitive biochemical tests for use with the aerobic and facultative bacteria has greatly influenced the speed with which these bacteria may be identified. The main emphasis in the past has been directed toward those bacteria belonging to the family Enterobacteriaceae, as evidenced by the many kits which have become available commercially during the past few years (1,2,9,10,13,14,16). The present study was carried out to determine the feasibility of using rapid biochemical tests for the identification of anaerobic bacteria. MATERIALS AND METHODS All anaerobic bacteria used in the study were recent laboratory isolates saved in chopped meat broth at room temperature in the dark. All organisms had been previously identified by the Virginia Polytechnic Institute (VPI) methodology (6). There were 112 organisms used in the study consisting of 1 Arachnia, 10 Bacteroides fragilis, 10 Bacteroides species, 10 Bifidobacterium, 10 Clostridium perfringens, 10 Clostridium species, 10 Eubacterium, 6 Fusobacterium, 5 Lactobacillus, 10 Peptococcus, 10 Peptostreptococcus, 10 Propionibacterium, and 10 Veillonella. All prereduced media were purchased from Scott Laboratories (Fiskeville, R.I.) and were inoculated using the VPI anaerobic culture system (Bellco). Prior to testing, each strain was subcultured to a fresh blood agar plate (BAP) prepared with Trypticase soy agar, 5% sheep blood, and 1% hemin-vitamin K solution (Scott Laboratories). The plates were incubated anaerobically in a vented GasPak jar evacuated and filled three times with 90% CO, and 10% H.. If the culture was pure, a single well-isolated colony was picked and transferred to prereduced peptone yeast glucose (PYG) broth or chopped meat glucose broth. All further subculturing was carried out from this I Present address: Curriculum in Medical Laboratory Sciences, University of Illinois Medical Center, Chicago, Ill. 60612. broth. In the case of the rapid tests, a fresh BAP supplemented with hemin and vitamin K was inoculated and incubated anaerobically for 24 to 48 h and was the source of inoculum for all the rapid tests. In addition, about 10% of the positive tests were retested with inoculum taken from prereduced supplemented brain heart infusion agar (BHIA) plates with 5% sheep blood added, a roll streak tube, and a PYG broth culture which was 24 to 48 h old. The standard tests used followed the procedures recommended by VPI (6) and were inoculated with 4 drops of an actively growing culture from either PYG or chopped meat glucose broths. The rapid biochemical tests used were: nitrate reduction, indole production, hydrolysis of gelatin, esculin, and starch, and the ONPG test for measuring hydrolysis of o-nitrophenyl-,B-D-galactopyranoside (ONPG) by beta galactosidase. All media were dispensed into tubes (13 by 100 mm). The nitrate test was prepared by dissolving 0.9 g of nitrate broth (Difco) in 100 ml of distilled water. The broth was dispensed in 0.5-ml portions into borosilicate tubes and autoclaved. The tubes were stored at 4 C no longer than 2 weeks. Tryptone broth was prepared by dissolving 1.0 g of tryptone broth (Difco) in 100 ml of distilled water and dispensing in 0.5-ml portions. The tubes were autoclaved and frozen at -20 C until used. The starch substrate was prepared by suspending 0.05 g of soluble starch in 100 ml of physiological saline (0.85% NaCl). The suspension was autoclaved, and the resultant solution was dispensed in 0.5-ml portions and stored at 4 C until used. The ONPG broth was made according to the formula of Cowan and Steel (4), dispensed in 0.5-ml portions, and frozen at -20 C. Esculin hydrolysis was determined by using 0.5 ml of enterococcosel broth (Gibco). The test for gelatin hydrolysis was performed on pieces of undeveloped X-ray film which had been cut into small strips (approximately 0.5 by 5.0 mm). The strips were placed into a 0.5-ml saline suspension of the test organism. All tests were performed with a sterile loop and by scraping up growth from the surface of the agar medium and inoculating directly into the broth substrate. When inoculum was taken from PYG broth, 2 drops of a 24to 48-h culture were pipetted directly into the rapid test medium. All tests were incubated aerobically at 35 C and read within 4 h. Tests for gelatin hydrolysis which were negative after 4 h were reincubated and read after 24 h. Reduction of nitrate was detected by adding 1 drop of reagent A (0.8% sulfanilic acid in 5 N acetic acid) and one drop of reagent B (0.5% alpha-naphthylamine in 5 N acetic acid) to the nitrate broth. The development of a red or pink color was considered positive, whereas the absence of color was considered negative. All negative tests were rechecked by adding a pinch of zinc dust to the nitrate broth. The absence of a red or pink color after the addition of zinc was considered positive. Indole production was detected by adding 2 drops of Kovac reagent to the tryptone broth. A positive reaction was denoted by a deep pink color in the surface layer. Starch hydrolysis was detected by adding 1 drop of a 1:4 dilution of Gram iodine solution (Kopeloff modification) to the starch broth. The development of a deep blue to black color was indicative of no starch hydrolysis. Any change in color as compared to an uninoculated control was considered positive. Esculin hydrolysis was indicated by the development of a brown or black color in the substrate broth. No change in color was considered negative. The gelatin tests were observed for the removal of the green gelatin emulsion from the X-ray strips. In a positive test the gelatin is removed, exposing the blue transparent strip. In a negative test the strip remains green. The ONPG tests were observed for the development of a yellow color, which was considered positive. In a negative reaction the substrate remained colorless. PathoTec strips for nitrate, indole, esculin, urease, Voges-Proskauer (VP), and ONPG were tested with the same 112 organisms. The tests were performed according to the manufacturer's instructions for Enterobacteriaceae, including 4 h of incubation in an aerobic environment. Inoculum was taken from a 24to 48-h anaerobic BAP. About 10% of the tests were performed in triplicate with organisms from BHIA, a roll streak tube, and PYG broth. When the PYG broth was used, 0.3 ml of a 24to 48-h culture was transferred to a test tube (13 by 100 mm), and a PathoTec strip was added. To determine the effect of atmosphere on both the rapid biochemical tests and the PathoTec test strips, about 10% of those organisms giving a positive reaction for a given test were retested simultaneously under aerobic and anaerobic conditions. A double volume of substrate was inoculated with 24to 48-h growth from an anaerobic BAP. The suspension of bacteria and substrate was then divided equally between two test tubes, and one was incubated aerobically at 35 C and the other anaerobically in an evacuation jar (90% CO,, 10% H2) also at 35 C. All media and reagents were checked with positive and negative controls to ensure their accuracy. Table 1 shows the results of the rapid tests compared with the standard tests as performed according to the VPI method. There was a 99% correlation with the indole test. One Clostridium species was positive with the standard test but negative with the rapid test. There was a 97% correlation with the test for starch hydrolysis. One Eubacterium and two Lactobacillus were positive with the standard test and negative with the rapid test. Nitrate gave a 95% correlation; both strains of Bacteroides corrodens, all three strains of Eubacterium lentum, and one Veillonella were positive with the standard method but were negative with the rapid tests. The rapid esculin test showed an 86% correlation with the standard method. There were 14 organisms which were positive with the standard test and negative with the rapid test. These included one Bacteroides fragilis, four Bifidobacterium, one Clostridium perfringens, five Clostridium species, two Fusobacterium, and one Peptostreptococcus. Two Clostridium perfringens were positive with the rapid test but negative with the standard method. Gelatin had the lowest correlation (77%). However, there were 13 instances where the standard test gave a weak positive reaction, and the rapid test was negative. A weak positive reaction in the VPI system is defined as liquification in less than one-half the time required for an uninoculated control. A weak reaction of this nature is of questionable significance in the classification of anaerobic bacteria, and so if we were to eliminate these 13 organisms from our comparison then 86 of 99 organisms would be in agreement (87%). Of the remaining organisms, three Bacteroides fragilis, one Bacteroides species, and one Fusobacterium were positive with the rapid test but negative with the standard test. One Bacteroides species, six Clostridium perfringens, and one Clostridium species were positive with the standard test and negative with the rapid test. Three strains of Clostridium were positive within 4 h with the rapid gelatin test. The remaining positive reactions were obtained after 24 h of incubation. Table 2 shows the comparison between the PathoTec test strips and the standard method. Indole showed a 96% correlation; three species of Fusobacterium and one Clostridium species were positive with the standard method and negative with the PathoTec strips. Esculin showed an 89% agreement. There were eleven organisms, including four Clostridium species, one Clostridium perfringens, three Bifidobacterium, one Peptostreptococcus, and two Fusobacterium, which were positive with the standard test but negative with PathoTec. One Clostridium perfringens was positive with Pa-thoTec but negative with the standard method. There were 21 organisms which were falsely negative for nitrate reduction by the PathoTec strips. These included two Bacteroides corrodens, two Propionibacterium, four Veillonella, three Eubacterium lentum, and ten Clostridium perfringens. In addition to the indole, esculin, and nitrate strips, PathoTec strips for urease, VP, and ONPG were also tested. The urease and VP strips were negative for most of the organisms tested. One strain of Bacteroides corrodens was urease positive. The ONPG strips compared in every instance with the rapid ONPG tests used routinely in our laboratory. RESULTS Concerning the effect of atmosphere, only the nitrate test seemed to prefer an anaerobic environment. All six organisms which had been falsely negative with the rapid nitrate test when incubated aerobically were positive when the test was incubated anaerobically. Thirteen of the 21 organisms, which were falsely negative for nitrate reduction by the PathoTec strips when incubated aerobically, were positive under anaerobic conditions. Four strains of Clostridium perfringens, two Bacteroides corrodens, and two Eubacterium lentum remained negative even under anaerobic incubation. Atmosphere seemed to have no effect on the tests for esculin hydrolysis. The tests for indole, starch, gelatin, and ONPG all seemed to prefer an aerobic environment as measured by the intensity and speed of the color development. Inoculum taken from the BHIA seemed to work slightly better than inoculum taken from the BAP. However, inoculum taken from the roll streak tube or PYG broth was less satisfactory. DISCUSSION Rapid testing of anaerobic bacteria is not new and has been reported as far back as 1941 by Reed and Orr (12). Clarke and Cowan (3) used several species of Clostridium in their studies of rapid techniques in 1952. Kaufman and Weaver (8) also used several species of Clostridium in their report of a combined media for detection of gelatin hydrolysis and indole production. In 1972, Sutter and Carter (15) evaluated four indole-spot tests for use in anaerobic bacteriology. The results presented here show that rapid testing of anaerobic bacteria is possible, and in most cases can be carried out within 4 h under aerobic conditions. An exception to this rule may be the test for nitrate reductase. Our findings are consistent with others (11,17) who have reported that nitrate reductase seems to have a diminished activity when exposed to oxygen. On the other hand, indole production has been shown to decrease when facultative organisms are incubated anaerobically (7). Fay and Barry (5) found the opposite to be true when testing for indole production with the obligate anaerobes. The discrepancy with our results may be explained by the fact that in 28,1974 their system growth was allowed to occur under the anaerobic environment, and thus the increased production of indole may be attributed to an increase in cell mass rather than increased enzyme activity. In our system growth was unlikely, and thus the increased indole production under aerobic conditions can probably be attributed to an increased activity of the enzymes involved. There was a wide range of colors produced with the test for starch hydrolysis. For this reason each test was compared to a negative control. Various colors, from light blue to brown to yellow, were obtained upon addition of the diluted iodine solution. These colors represent various degrees of starch hydrolysis, but, for the purpose of comparison, any color deviation from the negative control was interpreted as a positive reaction. A special precaution might be mentioned concerning the test for gelatin hydrolysis. Prior to reading the test, the tubes containing the gelatin strips should be shaken vigorously to remove any loose gelatin which might be adhering to the strips. Ten of the 21 organisms which showed a false negative reaction with the PathoTec nitrate strips were classified as Clostridium perfringens. The false negatives encountered with these and other species of anaerobic bacteria such as Veillonella, Bacteroides corrodens, and Eubacterium lentum suggest that the sensitivity of these strips might have to be adjusted before the nitrate strips can be recommended for use with the anaerobic bacteria. The effect of inoculum size seems to be consistent with our experience in rapid testing of facultative bacteria. A larger inoculum produces a stronger reaction in a shorter period of time. It is important to note that at all times it is assumed that one is working with a pure culture. Whenever contamination is suspected, a single well-isolated colony must be picked to fresh media to initiate a pure culture. Inoculum taken from a BAP or BHIA generally produced the best results. However, in some instances better growth was achieved on BHIA, and in these instances a stronger reaction was elicited in the rapid test system from this medium. The poorer correlation resulting from the roll tube might be explained by the fact that it was generally difficult to scrape up growth from the roll tube. In the case of the PYG broth, it may be that a 24-to 48-h culture is too old and that enzyme activity has already begun to diminish due to the presence of acids and possibly toxic end products in the culture medium. Also, the color of the broth may disguise or otherwise compromise the color of the end product in the biochemical reaction. For example, inoculum from PYG broth could not be used to determine the ONPG reaction, since the deep color of the broth obscured the yellow color produced in a positive reaction. In summary, rapid biochemical testing may be a useful adjunct to the identification of anaerobic bacteria. The fact that these tests can be run under aerobic conditions within 4 h and without the use of elaborate equipment suggests their desirability over the present systems for biochemical testing.

v3-fos

2018-04-03T05:47:17.715Z

1974-05-01T00:00:00.000Z

22852459

s2

Inactivation of Dried Bacteria and Bacterial Spores by Means of Gamma Irradiation at High Temperatures Dried preparations with Streptococcus falecium, strain Aj, and spores of Bacillus sphaericus, strain C A, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodu-rans, strain R1, and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resist-ant sporeformer B. sphaericus, strain C IA, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment. It been suggested brass cylinder, which was surrounded by a heat jacket. The temperature in the center of the cylinder was mea- sured by an electrical thermocouple. The temperature varied around the setting with i 2 C. In experiments where heating was applied before or after irradiations, irradiations were carried out in ""Co-facility m, loaded with 4,500 curies. The dose rate in the center of this gamma cell was 400 krad/h. It has been suggested that combinations of heat and ionizing radiation might be useful for sterilization of interplanetary vessels and foodstuffs (5,7). Such combinations may act additively or synergistically in inactivation of microorganisms without correspondingly additive or synergistic effects in material damage. Items which do not tolerate a dry heat sterilization or a radiation sterilization may thus be sterilized by a combination of the two methods. The present work aims at an evaluation of gamma irradiation procedures combined with heat treatments for sterilization of medical equipment. We have applied the bacteriological standard preparations we normally use for control of radiation sterilization and dry heat sterilization of such equipment supplemented with some experiments on a few other radiationresistant or heat-resistant strains. MATERIALS AND METHODS The following bacterial strains were used in the experiments: Streptococcus faecium, strain A11, ( Test pieces with S. faecium, strain A11, were taken from batches of reference standards prepared for control of the microbiological efficiency of radiation sterilization plants (3). Briefly, these standard preparations are prepared as follows. Heavily inoculated 5% blood agar plates are incubated at 30 to 32 C for 3 to 4 days. The cultures are scraped off the plates and suspended in serum broth. Droplets (0.02 ml) of the suspension (containing around 10' viable units) are placed on polyethylene foil and dried overnight in a glove box at 30% relative humidity as it has been found that humidity levels above 50% lead to reduced radiation resistance of the test pieces (4). Each dried test piece is sealed into a double envelope of polyethylene foil. Viable counts are determined by means of classical dilution techniques, by using 5% blood agar plates and by incubation at 30 to 32 C for 3 to 4 days. Test pieces with M. radiodurans, strain R,, were prepared by similar procedures. The strain was cultivated on tryptone-glucose-yeast extract (TYG) agar, and the test preparations were dried at ambient humidity in a laminar air flow cabinet. Test pieces with spores of B. sphaericus, strain CIA, were taken from batches of spore monitors pre-pared for control of the microbiological efficiency of radiation sterilization plants (3). These test pieces are produced by procedures similar to those used for preparing test pieces with S. faecium, strain A,1. They are dried at ambient humidity. For experiments at 100 C the test pieces were removed from the polyethylene envelopes and transferred to small aluminum capsules. Test pieces with spores of B. subtilis were taken from batches prepared for control of sterilization by dry heat, formalin, and ethylene oxide (6). Briefly these standard preparations are prepared as follows. Plate cultures from agar plates incubated for 5 days at 37 C are scraped off the plates and suspended in physiological saline and then mixed with sterilized quartz sand. The mixture is immediately vacuumdried at a pressure of 2 to 4 mm Hg for 24 h, and finally homogenized in a mortar. Each test piece, which consists of 120 mg of this spore sand and contains 2 x 10' viable units, is wrapped in two layers of paper. Spores of B. globigii were used in two kinds of preparations: dried serum broth preparations prepared in the same way as test pieces with spores of B. sphaericus, strain C A, and sand preparations prepared in the same way as test pieces with spores of B. subtilis. Most of the gamma irradiations were carried out in "Co-facility II, a gamma cell, which at the time of the experiments was loaded with 600 curies. During the time period, where experiments were undertaken, the dose rate in the center of the irradiation chamber decreased from 75 krad/h to 64 krad/h. Irradiations under this condition will be referred to as 70 krad/h. The dose rate could be reduced by lead shieldings. Two shielding configurations were -used. In one of these configurations the dose rate was reduced by a factor of 2.8, and irradiations under this condition will be referred to as 25 krad/h. In another configuration the reduction factor was 4.45 or 16 krad/h. For simultaneous heating and irradiation test, pieces were placed in the gamma cell in a 2-cm-wide brass cylinder, which was surrounded by a heat jacket. The temperature in the center of the cylinder was measured by an electrical thermocouple. The temperature varied around the setting with i 2 C. In experiments where heating was applied before or after irradiations, irradiations were carried out in ""Co-facility m, loaded with 4,500 curies. The dose rate in the center of this gamma cell was 400 krad/h. RESULTS Radiation inactivation curves for dried serum broth preparations with S. faecium, strain A21, M. radiodurans, strain R., spores of B. sphaericus, strain C A, and spores of B. globigii irradiated at room temperature are seen in Fig. 1. Figure 2 presents radiation inactivation curves for the same preparations at 80 C and 25 krad/h. It will be seen that the inactivation curves for the two spore preparations were unchanged, but that the LD 99.99 value for the preparations with the two vegetative strains was reduced by about a factor four. (LD 99.99 is the radiation dose, or heating time, that reduces the surviving fraction to 10-4, in Fig. 2: 0.8 Mrad for S. faecium, strain A21.) When the standard preparations with the vegetative bacteria were kept at 80 C for 48 h the surviving fraction for both strains was higher than 10%1, SO the simultaneous application of dry heat at 80 C and gamma irradiation acted clearly synergistically in the experiments with these two vegetative strains. At 80 C and 70 krad/h, the LD 99.99 was 1.4 Mrad for S. faecium, strain A21, and 1.6 Mrad for M. radiodurans, strain R1. At 60 C and 70 krad/h, the LD 99.99 for strain A21 was 2.8 Mrad and for strain R, it was 3.4 Mrad. Table 1 presents LD 99.99 values for irradiation of the four kinds of spore preparations at room temperature and at 100 C. It can be seen that the radiation resistance of the test pieces with spores of B. subtilis, normally used for control of sterilization by dry heat, was the same at 100 C and room temperature at the three dose rates tested. The radiation resistance of test pieces with spores of B. sphaericus, strain C IA, normally used for control of radiation sterilization plants was lower at 100 C than at room temperature. At 70 krad/h the reduction was small; at 16 and 25 krad/h the resistance was reduced by about a factor of four to the level of resistance observed with the subtilis preparations. The radiation resistance of the two kinds of preparations with spores of B. globigii at 100 C was lower than the resistance of the preparations with spores of B. subtilis and B. sphaericus at the same irradiation conditions. The radiation resistance of spores of B. subtilis and B. globigii in sand preparations was close to the resistance reported for clean spores of these strains (2,7). The radiation resistance of the B. globigii spores in serum broth preparations at room temperature was within the range reported for similar preparations with spores of B. subtilis and B. globigii (1,2). A few experiments with application of heat (100 C) and irradiation (400 krad/h) separately were carried out with preparations of spores of B. sphaericus, C IA. The LD 99.99 for heat alone was 100 h. When spores were pre-irradiated with 0.6 or 1. Fig. 1 irradiated at 80 C with 25 krad/h. DISCUSSION In the experiments with irradiation at 80 C, the two vegetative strains had a reduced radiation resistance, whereas the spores of B. sphaericus and B. globigii in serum broth preparations had an unchanged response. There were no indications of the so-called paradoxical effect, which has been reported with spores of B. megaterium and Clostridium botulinum (5,8). In these reports the spores had a higher radiation resistance at 80 to 90 C than at room temperature. This paradoxical effect disappeared at 100 C where the radiation resistance dropped to below the level observed at room temperature. In cases where it is known that sporeformers are totally absent, it may be beneficial to increase the irradiation temperature to 80 C in order to reduce radiation doses, but if the occurrence of sporeformers cannot be excluded, there may be a limitation of any benefits by irradiation at this temperature. At 100 C the spore monitor for radiation sterilization B. sphaericus, strain CIA, was the most resistant at all conditions tested. This, however, does not indicate per se that these preparations can be regarded as ideal monitors for evaluation of any combination of radiation and heat for sterilization, as more suitable strains may be isolated from the relevant environments. However, the present data may be applied in a preliminary analysis aiming at an identification of combinations of radiation and heat which may warrant further consideration, excluding combinations where benefits are too small to warrant investments in microbiological research and technical development. Figure 3 represents an attempt to do such an analysis based on the data obtained with B. sphaericus, strain CIA, at 100 C. In this analysis we arbitrarily have demanded that any combination of heat and radiation, which may warrant further consideration, at least should reduce both radiation LD 99.99 values and heat LD 99.99 values with a factor of two. The ordinate in Fig. 3 is LD 99.99 in hours, the abscissa is the dose rate in krad per hour. In this scheme we have drawn a full line, which represents the LD 99.99 values one would obtain if radiation and heat acted additively. The experimental results obtained at the three dose rates tested are plotted for comparison. It will be seen that at 16 and 25 krad/h the combination of heat and radiation acted more effectively than a pure additive effect, and that the results at 70 krad/h correspond to an additive effect. It can be seen that the only set of experimental data, which was below both "half" lines, were the data obtained at 25 krad/h. At this point the LD 99.99 values for both heat and radiation were reduced with about a factor of three. At 16 krad/h the reduction in radiation LD 99.99 value was the same as at 25 krad/h, whereas the heat value was only reduced by a factor of two. At 70 krad/h the data were below the "half' line for heat, but above the line for radiation. It appears from this analysis that combinations of heat and irradiation, where halving of both sterilization time and dose may be expected, are concentrated to a narrow dose-rate interval, approximately 20 to 40 krad/h. It appears unlikely that sterilization times and doses may be reduced by more than a factor of three at any combinations of 100 C heat and irradiation. Technological and economic considerations must decide whether the possibility of introducing this combination method in medical sterilization can justify the amount of microbiological work that will be necessary in order to ensure the general microbiological safety of the process. In view of the data presented here the sterilization times and doses suggested by Reynolds and Garst (7) for sterilization of spacecraft appear to be about one order of magnitude too low for sterilization of equipment for medical use. Although they use a slightly higher temperature (105 C), their suggestions of 132 krad at 12 krad/h or 252 krad at 36 krad/h, for example, as possible sterilization process parameters appear to be very optimistic. With any of the four kinds of spore preparations we have tested at 100 C, irradiation with 250 krad gave inactivation factors smaller than 10'.

v3-fos

2020-12-10T09:04:22.721Z

1974-12-01T00:00:00.000Z

237230797

s2

Arthrobacter globiformis and Its Bacteriophage in Soil Bacteriophages in soil for Arthrobacter globiformis were rarely detected unless the soil was nutritionally amended and incubated. In amended soil, phage were continuously produced for at least 48 h, and this did not require the addition of host cells. Rod and spheroid stage host cells added to the amended soil encountered indigenous bacteriophage, but added phage did not encounter sensitive indigenous host cells for some time, if at all. The indigenous phage in nonincubated soil seemed to be present in a masked state which was not merely a loose physical adsorption to soil materials but required growth conditions other than lysogeny for them to increase their titers. The possibility is discussed that the indigenous host cells in nonamended soil are present in a nonsensitive spheroid state, with the cells becoming sensitive to the phage in a rate-limiting fashion as nonsynchronous outgrowth occurs for a portion of the spheroid cells. Bacteriophages in soil for Arthrobacter globiformis were rarely detected unless the soil was nutritionally amended and incubated. In amended soil, phage were continuously produced for at least 48 h, and this did not require the addition of host cells. Rod and spheroid stage host cells added to the amended soil encountered indigenous bacteriophage, but added phage did not encounter sensitive indigenous host cells for some time, if at all. The indigenous phage in nonincubated soil seemed to be present in a masked state which was not merely a loose physical adsorption to soil materials but required growth conditions other than lysogeny for them to increase their titers. The possibility is discussed that the indigenous host cells in nonamended soil are present in a nonsensitive spheroid state, with the cells becoming sensitive to the phage in a rate-limiting fashion as nonsynchronous outgrowth occurs for a portion of the spheroid cells. Soil serves as a ready source for the isolation of virulent bacteriophage for many different bacteria (1). Little is known, however, about how these bacteriophage survive in soil and of the manner in which they locate and interact with their hosts in this habitat. Nor is it known how this interaction in soil is affected by pleomorphic growth cycles and dormancy states of the host bacteria. Answers to these obviously would be of interest from an ecological viewpoint, but, in addition, these answers might allow studies to be accomplished on the activities of specific bacteria in nature without isolating the bacteria. Thus, the specific bacteriophages for the bacterium are assumed to be present and operating in a defined manner in relation to their hosts, and these bacteriophages can easily be separated from the microbes and soil debris of the habitat and enumerated and evaluated by plaquing on host lawn plates of a specific bacterial species in the laboratory. The bacteriophage would thus serve as a natural internal indicator for the activities of a given bacterium in nature. Arthrobacter globiformis was chosen as the model bacterium for this study. It occurs naturally in soil (4) and, at least in the laboratory, it demonstrates a defined but not overly complex growth cycle (3,6,10,12,13). In addition, soil is known to harbor bacteriophages for this species (5,7,8,10). This bacterium and its bacteriophages were used, therefore, in the present ' This research was authorized for publication as paper no. 4746 in the journal series of the Pennsylvania Agricultural Experiment Station. study in an attempt to find answers to some of the more basic questions outlined above. MATERIALS AND METHODS Soil sample. A local Hagerstown silty clay loam (pH 6.0; plate count, 4.0 x 106/g at the time of use; moisture content, 25%; organic content, 3.5%; ref. 11) was used in this study. This sample consisted of the top 15 cm of soil collected directly beneath the surface vegetation, and it was stored in bulk for approximately 1 year in a sealed polyethylene bag. Media. Most experiments used BEG broth, which contained 0.3% beef extract and 0.5% glucose. The compositions of additional media are listed in Table 2. All media components other than sugars and inorganic salts were Difco products (Difco Laboratories, Detroit, Mich.). The synthetic medium included in Table 2 was based on Lochhead and Thexton (9) and contained glucose, 1.0 g; KHPO4, 1.0 g; KNOI, 0.3 g; MgSO4 7H2O, 0.2 g; CaCl2, 0.1 g; NaCl, 0.1 g; FeCl, .6HIO, 0.01 g; yeast extract, 1.0 g; and distilled water, 1,000 ml. The basal layer of phage-plaquing plates was BEG medium containing 1.5% agar; the overlay for these plates was the same medium but with 1.0% agar. A. globiformis cultures. The A. globiformis strains used in this study were American Type Culture Collection (ATCC) strains 8010 and 4336. Stock cultures were maintained on BEG medium containing 1.5% agar. Cultures of rodand spheroid-stage cells of this bacterium were grown by inoculating a loop of cells from a slant into 50 ml of BEG broth in a 300-ml baffle-bottom Erlenmeyer flask (Bellco Glass Inc., Vineland, N.J.) and incubating by shaking at 28 C. Cells harvested at 1 day were designated rod-stage cells, and those harvested at 3 days were designated spheroid-stage cells. These cells were washed by centrifugation and resuspended to their original volume 951 in distilled water for addition to soil, which was to be incubated at 60% of moisture-holding capacity (MHC), but were not washed or concentrated when used in broth studies. The lysogenic strain of 8010 occurred originally as a turbid plaque when a filtrate from a soil broth enrichment without added cells was plaqued with strain 8010 on nutrient agar. A culture recovered from this plaque was purified and then handled in a manner similar to that for the nonlysogenic strain. Soil incubation at 60% MHC. Portions (5 g) of soil in screw-cap tubes (18 [inner diameter] by 150 mm) were adjusted to 60% of the soil's MHC by adding distilled water plus a washed strain 8010 cell suspension (1.7 x 106 colony-forming units [CFUV5 g of soil) and/or a carbon or nitrogen solution nutritional amendment. The tubes were incubated with caps loose at 28 C for periods of from 0 to 4 days, and then the soil from each tube was added to 100 ml of BEG broth in a 300-ml baffle-bottom flask. This was shaken for 10 min and then centrifuged at low speed. The supernatant fluid, containing the phage, was passed through a membrane filter (0.3-Am pore size; Millipore Corp., Bedford, Mass.) and then further diluted in sterile BEG broth for phage plaquing. Broth-soil phage enrichments. Soil (4 g) was added to 100 ml of broth medium in a 300-ml baffle-bottom flask. In some experiments the flasks also received unwashed A. globiformis cells, either rod or spheroid stage, at a final concentration in the flask of 1.7 x 107 or 1.4 x 107 CFU/ml, respectively; the final flask titer of added FX-1 phage was 40 plaqueforming units (PFU)/ml and of added soil-mixed phage preparation was 100 PFU/ml. These flasks were shaken at 28 C, and, at various time intervals, 5-ml samples were removed and subjected to low-speed centrifugation. The supernatant fluid was then passed through a 0.3-,um membrane filter, and the filtrate was diluted in BEG broth for phage plaque assay. Two or more flasks per treatment were included so that an overall large withdrawal of fluid volume from any one flask during sampling would not occur to affect the results. Cell-phage interaction in absence of soil. To 80 ml of broth medium in a 500-ml Klett side-arm Erlenmeyer flask (Bellco Glass Inc., Vineland, N.J.) was added rodor spheroid-stage unwashed cells at a final concentration in the flask of 106 CFU/ml; the soil-mixed phage preparation, when added, was at a final titer of 50 PFU/ml. These flasks were shaken, and, at various time intervals, turbidity was measured as Klett units and 5 ml-samples were withdrawn and treated as above for phage plaque assay. For sonic treatment trials, phage suspension (1 ml), strain 8010 cell suspension (1 ml), soil (4 g), or mixtures of these were added to 100 ml of sterile BEG broth, and this was sonically treated for either 1 or 12 min in a Biosonic I oscillator (Bronwill Scientific, Inc., Rochester, N.Y.) operating at 112 W accoustic energy at the probe tip. The probe had been sterilized with alcohol. Phage sources and assay. Most phage samples for plaque assay were diluted in BEG broth. One milliliter of a dilution to be plaqued and 0.15 ml of strain 8010 broth culture (rod stage) were added to 2.5 ml of BEG 1% agar medium, and this was applied as an overlay on a BEG 1.5% agar basal layer. The experiments reported in Table 2, however, used nutrient broth and agar, respectively, for dilution and plaquing. The plates were incubated 48 h at 28 C, and then the plaques were counted. The plates were again observed at 4 days of incubation so as to note the occurrence of possible plaques not present at 2 days and whether any plaques had markedly increased in size. Periodic electron microscopy checks of phosphotungstic acid-negative stains were made of plaques resulting from soil enrichments to be sure that bacteriophage actually were causing the plaques. Virulent phage strain FX-1 was recovered from a soil enrichment (without added host cells) that had been plaqued on strain 8010 on nutrient agar. It was purified on this medium, and phage preparations were prepared by suspending the surface agar from confluent lysis plates in nutrient broth. The residual cells and agar were removed by low-speed centrifugation, and the phage suspension was passed through a sterile 0.3-aum membrane filter and refrigerated. The soil-mixed phage preparation was made as follows. Soil (12 g) was added to 300 ml of BEG broth in a 1,000-ml Erlenmayer flask. This was shaken for 24 h at 28 C and then clarified by low-speed centrifugation. The supernatant fluid was sequentially passed through sterile 0.8-and 0.3-am membrane filters to yield a mixed phage suspension containing 10' PFU/ml as plaqued on strain 8010. Phosphotungstic acid-negative stains of this preparation viewed by electron microscopy showed phage of several different morphologies, but no bacterial or other cells. RESULTS Soil present. We have not been successful in extracting bacteriophage for A. globiformis ATCC 8010 from our soil without first incubating the soil. Adjustment of the soil to 60% of its MHC with distilled water, with or without rod-stage host cell additions, and incubating at 28 C gave a few phage in one instance but none in the second instance (Table 1). Usually, however, no phage could be detected under these conditions. In contrast, incubation of soil amended with glucose or sucrose provided extensive phage production for this bacterium, and the response was greater when washed rod-stage host cells had been initially added to the soil. Several broth media were evaluated with the object of finding one that would more easily allow study of the phage enrichment process when host cells had not been added than was possible by incubation of the soil at 60% MHC. In addition, it was desired that the soil with its indigenous host cells and phage be, dispersed and agitated in a liquid medium, so that the effects of spatial discontinuities separating the phage from their hosts would be lessened. A Separate but similar experiment using 25 g of soil in polyethylene-covered glass tumbler. d Starch, glucose, and sucrose added at 50 mg/5 g of soil. e (NH4),SO4, NaNO., and urea added at 5 mg/5 g of soil. medium containing 0.3% beef extract and 0.5% glucose (i.e., BEG medium), inoculated and shaken with 4 g of soil (host cells not added), yielded 2.7 x 10' PFU/ml at 3 days of enrichment (Table 2). This medium, therefore, was used in most of the succeeding trials. BEG broths were inoculated with 4 g of soil, with and without additions of host cells or phage, and shaken 1 to 2 days at 28 C. The soil not receiving added phage or host cells continuously produced phage over the time periods tested ( Fig. 1 and 2). The added soil-mixed phage preparation, however, apparently encountered few if any sensitive indigenous host cells in the soil, and a delay in addition of the phage until 6 h of incubation had occurred did not change this picture. The pure laboratory strain of virulent phage (FX-1) did encounter some sensitive indigenous host cells in the soil (Fig. 2), but not until sometime between 14 and 24 h of incubation; this was shown to be a repeatable phenomenon. This in situ phage production by FX-1 was sensitive to sonic treatment. A 1-min sonic treatment at zero time of the broth containing the soil and FX-1 virtually eliminated this response. In contrast, the indigenous phage production occurring in the soil enrichment without added phage or host cells withstood a 12-min sonic treatment. Rod-stage cells of A. globiformis ATCC strains 8010 and 4336 responded alike in phage production when they were added to soil enrichments (Fig. 2). Note that the soil incubated with strain 4336 was plaqued on strain 8010. Both bacterial strains vigorously produced phage starting at about 8 h and extending to about 14 h of incubation. In contrast, the added spheroid cells of strain 8010 delayed phage production during the first 9 h (rate compared to the soil-only control; Fig. 1), but then continuously produced phage at least through 24 h of incubation. When the soil-mixed phage preparation and rod-stage host cells of 8010 were added simultaneously to the soil and the soil was incubated, the phage production curve closely resembled that obtained when only the bacteria were added without adding phage (see Fig. 1 and 2). Thus, in this case, the actual source of the phage inoculum (whether added or indigenous) producing the additional phage was not clear. VOL 28, 1974 being liberated in the soil as a result of growthrelated processes or simply were being physi-7.0 cally desorbed from a masked state on the soil debris, flasks containing distilled water or broth medium were incubated at 4 C, and filtrates prepared at intervals during incubation were 6.0 -enriched for 24 h at 28 C, as above, with a pure culture of strain 8010. The cold-temperature soil incubation comprised, sequentially, shaking for 13 To determine whether the phage were cells plus soil, genic strains occurred in heart infusion broth, and the growth of both strains was equivalent in this medium. The delay for growth initiation by the spheroid-stage cells was still present, however, in this medium. Figure 5 presents growth results and phage production resulting from lysogeny when lysogenic strain, spheroid-stage cells were used as inoculum; rod-stage cells were not tested for phage production. Addition of the h. These results for the rod-stage cells approximately correspond to those for rod-stage cells in the presence of soil ( Fig. 1 and 2), but the initiation of phage release for the spheroid-stage cells in Fig. 3 (absence of soil) seems to be delayed approximately 3 to 5 h as compared with the results in the presence of soil (Fig. 1). In the absence of added phage, both rodand spheroid-stage cells of the lysogenic strain produced almost no phage in this broth medium; 12 PFU/ml were detected at 18 h for the rod-stage cells, and 30 PFU/ml at 12 h for the spheroidstage cells. At other incubation times only 1 or 2 PFU/ml were detected. Both the lysogenic and nonlysogenic strains grew at a similar rate in this medium; Fig. 4 shows growth for rod-and spheroid-stage cell inocula of the nonlysogenic strain. As compared to BEG broth, considerably more growth of both the lysogenic and nonlyso- mixed-soil phage preparation did not materially change this picture for either growth or phage production. In contrast to these results, the spheroid-stage cells of the nonlysogenic strain (rod cells not tested) were virtually immune to added phage in the heart infusion broth. Growth as measured by turbidity was not altered, and the added phage were barely detectable or not detectable in samples taken during incubation. Both the rodand spheroid-stage cells of strain 8010 were quite resistant to sonic treatment; the CFU per milliliter were identical for cells not sonically treated and cells sonically treated for 12 min. The phage, however, displayed some sensitivity towards sonic treatment. Phage strain FX-1 showed 97% survival of PFU per milliliter with 1 min of sonic treatment and 43% with 12 min; the respective survival values for the soil-mixed phage preparation were 84 and 50%. Enrichment in shaken culture was not involved in these determinations, and soil was not present. The nonsonically treated, soil-mixed phage preparation was relatively resistant to the shaking involved in broth incubations; shaking in BEG broth in the absence of host cells or soil gave percent survivals of the phage at 5, 12, and 24 h, respectively, of 60, 35, and 31%. Rod-and spheroid-stage cells. Rodand spheroid-stage strain 8010 cells were used as inocula in these studies. The rod cells were from near the end of the logarithmic growth phase, and they presented a Klett value of approximately 256 units and a plate count of 1.7 x 109/ml. The respective values for the spheroid cells, which received two additional days of incubation, were similar, being 226 units and 1.4 x 109/ml. In addition to this age disparity, these cells differed in their morphology. The rod cells were short, gram-negative rods surrounded by what appears to be a slime layer. The spheroid cells were gram-positive coccoid rods slightly pointed at each end and surrounded by a thin, gram-negative casing. The delay in growth initiation observed when the latter cells were used as inoculum (see Fig. 4) seems to be due to the time required for pleomorphic outgrowth of these cells. The outgrowth at 12 h is shown in Fig. 6, which corresponds to a point on the growth curve (Fig. 4) when growth increase is not yet measurable as turbidity. DISCUSSION Bacteriophages for A. globiformis ATCC strain 8010 were rarely detected in our soil unless the soil was amended nutritionally and incubated. This was true for soil incubated with water at 60% of the soil's MHC and for soil shaken in water. Addition of washed host cells did not change this picture. In contrast, addition of glucose or sucrose to soil at 60% of MHC, with or without a simultaneous addition of host cells, stimulated a bacteriophage titer buildup to approximately 104 to 106 PFU/g of soil by the second day of incubation. Amendment of the soil with nitrogen-containing compounds, however, was less stimulatory to the production of phage. The sequence of events and the activities of the phage and host cells occurring during soil incubation were more easily studied by using shaken-aerated broth enrichments with soil, and, for approximately the first 24 h, these enrichments seemed to be representative of the 60% MHC soil incubations. Evaluation of several broth media showed that a medium comprised of 0.3% beef extract and 0.5% glucose (i.e., BEG broth) provided acceptable phage titers in the shaken soils as plaqued with A. globiformis ATCC strain 8010. When this medium was used for growing strain 8010 in the absence of soil, a reasonable growth rate and total amount of growth were obtained, although these were less than those obtained with a nutritionally richer medium such as heart infusion broth. Soil shaken in BEG broth (host cells or phage not added) started to produce phage which plaqued on strain 8010 at about 6 h of incubation, and this phage production continued for 48 h or longer. However, a decrease in the phage production rate usually occurred by about 24 h (Fig. 2). Host cells of strains 8010 and 4336 added to this soil enrichment encountered bacteriophage, as shown by phage titers greater than with the soil alone, starting at about 8 to 9 h of incubation. In contrast, phage added to the soil (host cells not added) did not seem to encounter sensitive host cells for some time, if at all. Laboratory strain FX-1 lytic phage interacted with the indigenous host cells in the soil starting some time after 14 h of incubation ( Fig. 2; rate compared with the soilonly control), but a soil-mixed phage preparation that had originally been recovered from this soil did not find sensitive host cells (Fig. 1). The reaction of the indigenous host cells in soil to the added FX-1 phage seemed to be a different phenomenon from that of the interaction of the indigenous phage and indigenous host cells already in the soil. Thus, sonic treatment of the mixture of soil and phage FX-1 before incubation destroyed the phage production response to this phage in the soil, but the phage production by the indigenous phage and were quite resistant to the sonic treatment levels used in this study, and that both the FX-1 phage strain and the soil-mixed phage preparation were relatively resistant to sonic treatment. The phage production in soil not receiving added host cells or phage did not seem to be merely a physical release of phage which had been masked by adsorption to soil materials, because phage titers did not build up in the soil when it was incubated in the cold. In addition, sonic treatment of the soil did not release additional phage over those in nonsonically treated soil. The strain 8010 cells used in this study were either gram-negative, short rods (rod-stage cells) taken near the end of their logarithmic growth or gram-positive spheroids (spheroidstage cells) from late in the maximal stationary phase of growth. On inoculation into fresh BEG medium, the latter cells delayed growth for approximately 6 h longer than did the rod cells. This extra time was required fdr-pleomorphic outgrowth of the spheroids (Fig. 6) before initiating a rapid growth phase. A lysogenic strain of 8010 was also used in these studies and, as concerns spheroid pleomorphic outgrowth and growth of the rod cells, it behaved in a manner similar to that of the nonlysogenic strain. The choice of bacterial cell numbers (either rod or spheroid stage, lysogenic or nonlysogenic) to be added to the soil or used as inoculum in pure culture studies was quite empirical because, although Arthrobacter species cells are thought to be quite numerous in soil (2), it was not known how many A. globiformis-like cells might be in soil that could respond to phage that plaque on strain 8010. Likewise, the numbers of phage PFU to be added to soil or pure bacterial cultures were open to question. Since the phage numbers recoverable from soil were nil or very low unless incubation with nutrients was employed, it was decided to use a low ratio of PFU to CFU in the pure culture experiments. The final ratio when soil was not present was approximately 1 PFU/2 x 10' CFU. A comparison of Fig. 3 and 4 for pure cultures in the absence of soil shows that, for both rodand spheroid-stage cells used as inoculum, phage production is initiated as the rod cells start to multiply and is rapid thereafter. The above-mentioned delay for spheroid pleomorphic outgrowth thus also applies as a delay for phage production. Figure 3, in addition, shows that the lysogenic strain in BEG broth with added phage responds with growth and phage production in a manner similar to that of the nonlysogenic strain. The delay for phage production by indigenous soil phage acting on spheroid-stage cells added to the soil, and the shorter delay for added rod-stage cells, are shown in Fig. 1. After these delays, however, phage production in both cases is rapid, with rates roughly equivalent to those resulting when 100 PFU of the soil-mixed prepa-ration per ml are reacted with 106 CFU of strain 8010 per ml (Fig. 3). These rates are considerably greater, however, than those for the interaction of indigenous phage with indigenous host cells ( Fig. 1 and 2). The latter rates could indicate that the sensitive host cell level naturally present in soil actually is quite low. From this viewpoint it is of interest that the 24-h indigenous phage production titers in Fig. 1 and 2 (host cells and phage not added) can be duplicated (experiment not reported) by shaking a mixture of 80 CFU of strain 8010 rod-stage cells per ml and 250 PFU of the soil-mixed phage preparation per ml for 24 h in BEG broth. A low available host cell number could also explain the inability of the added soil-mixed phage preparation to locate sensitive host cells, and of added strain FX-1 phage to find sensitive host cells until some time after 14 h of incubation. A low sensitive host cell level, however, does not necessarily mean that the total numbers of host cells are low. It is generally assumed that Arthrobacter species occur naturally in soil in the spheroid (coccoid) stage (4) and, although the present study has shown a defined time requirement for spheroid outgrowth, nothing is known about the frequency with which this outgrowth occurs in nature. In other words, the proportion of the spheroid population that does not respond with outgrowth to a given environmental stimulus is not known. A nonsynchronous spheroid outgrowth in nature, however, could provide a continuous but ratelimiting source of low numbers of cells sensitive to phage and, thus, might explain the present results. The survival of this bacterium in nature thus could well depend on there being a reservoir of spheroid cells which do not undergo outgrowth even though growth conditions may have improved. An alternate explanation for our results would be that the phage production in soil observed when neither host cells nor phage were added represents in total the results of lysogeny for the in situ cells or, alternatively, an initial production of phage through lysogeny with these phage then acting in a virulent manner on other sensitive host cells. These do not seem to apply in our study, and this conclusion is based on the opposing results obtained with the use of BEG and heart infusion broths, and on the fact that lysogeny as a means of inoculating sensitive host cells would impose too long a time delay before phage production could be initiated by the latter cells. Soil incubated in heart infusion broth (host cells and phage not added) produced only barely detectable levels of phage as contrasted with the phage yields produced in BEG broth (Table 2; Fig. 1 and 2). When soil was not present, the lysogenic strain of 8010 without addition of phage produced easily detectable levels of phage in heart infusion broth (Fig. 5), but barely detectable levels or no phage in BEG broth. Phage production in soil thus more closely resembles that associated with the nonlysogenic strain. Obviously, however, we used only one lysogenic strain in this study and, of course, do not know whether the soil harbors other lysogenic strains that would act in an entirely different manner under our experimental conditions. Based on the foregoing discussions taken as a whole, it would seem that in non-nutritionally amended soil the numbers of naturally occurring bacteriophage capable of plaquing on A. globiformis ATCC 8010 are low but not nil. Their numbers, however, cannot be precisely quantified, because they appear to be masked in some manner, other than through lysogeny or a loose physical adsorption to soil materials, so that they cannot be washed from the soil but, still, are available with the proper time delays for reaction with added rodor spheroid-stage host cells. The naturally occurring host cells in soil for these phage either are present in very low numbers or are present in a form insensitive to the phage. In the latter case, incubation with added nutrients would nonsynchronously change them into a sensitive state so that at any one time only a portion of the cells could interact with the phage.

v3-fos

2020-12-10T09:04:20.704Z

1974-10-01T00:00:00.000Z

237231549

s2

Identification of Major High-Boiling Volatile Compounds Produced During Refrigerated Storage of Haddock Fillets The two major high-boiling volatile compounds produced during refrigerated storage of haddock fillets were found by gas chromatography and mass spectroscopy to be phenethyl alcohol and phenol. The major genera of bacteria responsible for the spoilage of refrigerated fish have been studied extensively. Little work, however, has been reported on the major high-boiling metabolites produced in fish tissue during refrigerated storage. Wong et al. (3) reported the formation of benzene, toluene, and several ketones in cod held at 0 C. This study documents the formation of phenethyl alcohol and phenol as the major high-boiling volatile components produced in haddock fillets stored at 2 C. MATERIALS AND METHODS Isolation of volatile components from haddock. A 30-g amount of fish tissue was placed into a stainless-steel centrifuge tube (50 ml capacity), 15 ml of Mazola corn oil was added, and the mixture was homogenized with a glass rod. After thoroughly blending in this manner, the oil phase was separated by centrifugation at 17,000 x g for 20 min. A 10-ml volume of the oil was then used for molecular distillation (2). The sample container was immersed in an oil bath at 80 C, and the oil sample was stirred continuously by a Teflon-coated magnetic stirring bar. The volatiles were collected from the oil by distillation under a vacuum of approximately 10-3 torr, and condensed onto a liquid nitrogen-cooled cold finger (2). After 2 h of distillation, the cold finger was removed, and about 10 ml of anhydrous diethyl ether was used to rinse the collecting surface. Prior to gas chromatography analysis, the ether was concentrated to 20 Mliters in a vial with a gentle flow of nitrogen. A volume of 2 Mliters was used for injection into gas chromatography columns. Separation and identification of the major volatile components. A Perkin-Elmer model 881 dualcolumn gas chromatograph equipped with a flame ionization detector was used with a 1 mV full-scale Honeywell model W recorder for analysis of collected volatiles. After various columns were tested, a 12-ft (ca. 3.7 m) column packed with Carbowax 20M was used for initial separation of the volatile components. I Paper no. 1007 of the Massachusetts Agricultural Experiment Station, University of Massachusetts at Amherst. Column temperature was maintained at 145 C, and prepurified nitrogen (Airco) was used as the carrier gas. Using a combined gas chromatograph and mass spectrometer unit, the mass spectra of the major high-boiling volatile components were obtained. The effluent from an Aerograph 1200 gas chromatograph was admitted via a heated line to a Bieman Helium separator and then to the ion source of an Hitachi-Perkin Elmer RMU-6A mass spectrometer. The identification of volatile compounds was verified by comparing their mass spectra and gas chromatography retention times on two different 12-foot columns (Carbowax 20M and diethylene glycol succinate). Bacterial count on haddock fillet. The viable bacterial count on the haddock fillet used above was determined on pour plates of Trypticase soy agar without dextrose (BBL) incubated at 2 C for 6 days. RESULTS AND DISCUSSION Increase of volatile components during refrigerated storage of haddock. A market fresh haddock fillet of high organoleptic quality was found to contain few high-boiling volatile compounds at zero time storage (Fig. 1). After 5 days of storage, a number of high-boiling volatile peaks were present in relatively low concentration. On day 9 of storage, a major peak, designated peak 40, was present. On day 15, coincident with the development of a maximal bacterial population on the tissue (Fig. 2), peak 40 had decreased notably and a third peak, designated peak 44, predominated. On day 20 of storage, peak 40 was only barely detectable, whereas peak 44 had greatly increased. Identification of the major high-boiling volatile compounds. Peaks 40 and 44 were identified by mass spectroscopy as phenethyl alcohol and phenol, respectively ( Fig. 3 and 4). Authentic phenethyl alcohol (C6H5CH2CH2OH, Eastman Organic Chemicals) and phenol (Fisher Scientific Co.) were used for verification. Both the mass spectral data and gas glycol succinate column. Phenol had a retention time of 49 min on the Carbowax column and 13 min on the diethylene glycol succinate column. The genus Achromobacter was found to be responsible for phenethyl alcohol production and is the subject of an accompanying report (1).

v3-fos

2014-10-01T00:00:00.000Z

1974-05-01T00:00:00.000Z

18448469

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Lead content of foodstuffs. The lead content of a number of foodstuffs, particularly baby fruit juices and milk, is reported. Samples were analyzed in quadruplicate by using an automated Delves cup atomic absorption procedure. A large proportion of the products examined contained significant amounts of lead. Of 256 metal can examined, the contents of 62% contained a lead level of 100 μg/l. or more, 37% contained 200 μg/l. or more and 12% contained 400 μg/l. lead or more. Of products in glass and aluminum containers, only 1% had lead levels in excess of 200 μg/l. Lead levels of contents also correlate with the seam length/volume ratio of the leaded seam can. A survey of bulk milk showed a mean lead level of 40 μg/l. for 270 samples; for canned evaporated milk the mean level was 202 μg/l. These data indicate a potential health hazard. Introduction Lead poisoning is an important public health problem. At present, over 250,000 children per year in the United States are screened for undue absorption of lead. Screening is concentrated in decaying urban areas, where children have access to chipped and peeling paint containing up to approximately 20 %o lead. Since the Industrial Revolution and particularly since the mass introduction of automobiles around 1950, environmental lead levels have markedly increased. Lead is found in drinking water, canned fruit, vegetation growing beside roads, toothpaste, air particulates, dirt, pencils, cigarette ash, newsprint, putty, and numerous other materials in everyday use (1). In December 1972 we carried out a limited survey of the lead content of a number of foodstuffs and found significant levels in some canned fruit juices. This prompted a more thorough survey of the lead content of *Division of Laboratories and Research, New York State Department of Health, New Scotland Avenue, Albany, N.Y. 12201. canned and bottled fruit juices, canned and bulk milk products, beverages, and some canned fruits. Our findings are reported below. A further survey of canned milk products carried out in September 1973 is also included. Experimental Procedures Samples All nondairy foodstuffs were purchased in Albany County, New York, during May 1973, with the exception of a small amount of canned goods processed for institutional use (obtained from the New York State Department of Mental Hygiene). Bulk milk samples were obtained from a large number of processing plants at various locations in upper New York State, and canned milk products were purchased in Albany County during September 1973. Instrumentation The instrumental system has been described in a previous communication from this laboratory (2). Briefly, it comprises a single-beam atomic absorption spectrometer built up from a hollow cathode lamp with dc power supply, a burner-Delves cup injector, an f/3.5 monochromator, a photomultiplier, a photon counter, an interface, and a computer. Insertion of the cup into the flame triggers a delay. The system then measures the intensity of transmitted radiation for 200 successive 30-msec periods before, during, and after the absorption peak. Intensity data are converted to absorbance, and the integrated absorbance for the transient lead atomization process is computed. The peak absorbance value is also measured and printed out. The following instrumental settings were used: slit height 15 mm, slit width 150 n, lamp current 8 mA, wavelength 217.0 nm. A slightly fuel-rich air-acetylene flame was used, with the cup lip ca. 2 mm below the entrance hole in the ceramic absorption tube. The instrument is initially calibrated with standards made up from spiked milk. This matrix was chosen because, unlike aqueous lead standards, it gives sharp peaks for the lead-specific absorption. The measurement time base can be identical for the standards and the samples, since they both contain organic matter which enhances the rate of vaporization from the cups into the flame gases. Standards of 800, 600, 400, 200, and 0 jg/l. of added lead were used for all calibration. The milk used for these standards had been previously found to contain only low lead levels by a solvent-extraction macroprocedure. The computer program allows the operator to select a calibration procedure in which four sets of five standards are introduced into the system. The data from these standards are used to construct a least-squares calibration line. If the data points give a good coefficient of correlation (R .0.98) to the calculated curve, the instrument is ready for analysis of unknown samples. A correlation coefficient <0.98 causes the system to ask for recalibration before samples can be run. Samples are run in quadruplicate; the average integrated absorbance is calculated; and the lead concentration, taken from the cali-bration curve, is stored by the computer. The total analysis time for one sample with four replicates is about 1 min. Sample Preparation For samples (juices, milk, etc.) which were sufficiently liquid to be pipetted with an Eppendorf after shaking, 50-pl portions were diluted with 4 parts of deionized water, and 50-4ul aliquots were pipetted into nickel Delves cups. These solutions were dried at 1400 C for 15 min and then introduced into the automated flame spectrometer. For more viscous products (purees, baby food, tomato paste, etc.), a twofold dilution with equal amounts of deionized water preceded this treatment to facilitate precise pipetting. For fruit samples, pickled produce, and other products where a solid was packed in a syrup or other fluid, no attempt was made to homogenize the solid; only the fluid was analyzed. Samples were also drawn from cans of viscous tomato paste as follows. A longneedled Cornwall syringe of 5 ml capacity was inserted to the bottom of the can in the appropriate sample area. (The can had been handled carefully prior to sampling to ensure that the contents did not mix or move.) A 1-ml sample was taken out as the syringe was withdrawn; this was assumed to prevent the contents from mixing due to turbulence within the can. The sample was then diluted with 2 ml of deionized water and prepared as above. Soldered can seams were analyzed for lead content by cutting a 1-cm portion of the seam length and dissolving the same in 25 ml of concentrated nitric acid making the solution finally to 100 ml with deionized water. This solution after a further 100-fold dilution was sprayed directly into an airacetylene flame in the normal manner to determine the lead level by conventional atomic absorption spectrometry. Results Lead Content in Foodstuffs Figure 1 shows the distribution of lead lev- els in a group of 254 different containers of which 205 were metal cans. In Figure 2 the distribution for the metal cans with soldered seams is compared to that for bottled products. A comparison of the same product in different containers was possible in only a few cases, and since we could not determine whether the contents came from the same factory and batch, a parallel comparison is not presented. However, the mean lead concentration of the canned products (167 jlg/l.) is obviously higher than that of the bottled products (42 /Ag/l.), suggesting the effect of packaging on the lead level of food products. Baby Foods Food products marketed as baby foods are generally packaged in small cans or bottles of 4-5 fl oz capacity. A group of 86 canned foods and 35 bottled products were analyzed. The results (Fig. 3) show a mean lead level for the canned foods of 202 1kg/l., with 37% of the cans having lead levels above 200 jug/l. In contrast, the bottled products (mainly puree dinners) had a mean lead concentration of 35 1tg/l., with only 1 (220 pg/l.) in excess of 100 #,g/l. Of the canned baby foods analyzed, a large proportion were fruit juices and fruit juice mixtures, all pH 2.7-3.9. This acidity, combined with a high seam/volume ratio, would explain the high lead contents. The seams of these cans, in common with those of the 100, dered-seam cans, 35% appeared to have had their inner surfaces lacquered at some stage of manufacture. In several instances the lacquer was discolored at the seam and appeared blistered, as if the lacquer had been sprayed on the metal prior to soldering. In nearly all cases of lacquering, some areas of solder were exposed to the contents of the can, especially at each end of the seams, where the solder had run while molten. To determine if the higher lead levels in canned goods might be a result of leaching of lead from the seam, we studied the distribution of lead in the contents of a series of cans of viscous tomato paste. The results for five 8-oz cans of tomato paste, sampled in five positions across a diameter passing through the seam, are shown in Figure 4. In all cases, position 1 (nearest the seam) shows a higher lead level than the opposite side of the can. Evidently the can contents were leaching lead from the soldered seam. This is especially likely with the tomato paste, which is highly acidic (pH -4.0). Figure 5 shows the data from the same cans after they had been stored open at room temperature for 24 hr. The lead distribution is even more indicative of leaching from the seam, and the lead levels (up to 5000 jg/l.) are becoming extremely high. If lead is entering the can contents via the seam, there should be a relationship between the seam length/can volume ratio and the lead level of the contents. The data from three groups of cans selected for seam length/volume ratios of 1.25, 0.75-1.25, and <0.75 confirm this assumption (Fig. 6). In the group with a high seam length/volume ratio, significantly more cans fall within the higher lead levels. In the group with low seam length/volume ratios, the majority are <100 tAg/l. A breakdown of the data by container type ( For a survey of lead levels in bulk fresh milk, 270 samples were obtained and analyzed in quadruplicate. The distribution (Fig. 7) indicates an average lead level of 40 jug/ 1., with no samples greater than 200 ,ig/l. The -lead levels in cows' milk may result from environmental contamination of feed and grass, especially if pasture is located near main highways. In addition to bulk fresh milk, 51 soldered seam cans of evaporated milk were analyzed. Moreover, the tops and bottoms had been soldered, and the hole used to fill each can had a solder button as a seal! Lead concentrations ranged from 10 Ag/l. up to 820 ALg/l. The mean lead level was 202 ug/l., and 55%o of the cans had concentrations >200 ,ug/l. These results are significantly higher than those obtained by Lamm and Rosen in a similar survey of canned milk products (3). Discussion Considerable quantities of lead were found in two classes of foodstuffs: canned baby fruit juices and canned milk products. Lead in fruit juices probably presents the greater health hazard, since there is evidence that calcium has a "protective" effect against lead toxicity (4). These lead sources may make a considerable contribution to the lead body burden for a young child. For example, if a child is fed canned products averaging 300 jLg lead/ 1. (50%7o above the mean lead level found in this study), it would require 1 liter per day to meet the maximum daily permissible intake (MDPI) recommended by an ad hoc committee of the Department of Health, Ed-ucation and Welfare (5). It would take only Q.33-0.67 I./day to exceed the MDPI recommended by tg/day) (6) and only 0.17 I./day to exceed the World Health Organization MDPI for a 10-kg child (5 JAg/kg body weight/day (7). These calculations probably understate the health hazard, since (a) they omit contributions from other lead sources, including paint, dirt, and air particulates; (b) children ingest these lead sources at a much earlier age than with paint ingestion; and (c) there is some evidence that very young children absorb lead more efficiently than older children (8).

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2020-12-10T09:04:12.497Z

1974-02-01T00:00:00.000Z

237230995

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Soil Ecology of Coccidioides immitis at Amerindian Middens in California Outbreaks of coccidioidomycosis and isolation of Coccidioides immitis have been reported from Amerindian middens. This study was undertaken to determine the most important ecological component(s) for the occurrence of C. immitis at archeological sites. Soils from 10 former Indian villages with no prior history of coccidioidal infection were collected and cultured. The physicochemical properties of the midden soils were compared with nonmidden soils and positive soils. The following theories for the sporadic distribution of the pathogen in the soil of the Lower Sonoran Life Zone were considered: (i) the Larrea tridentata (creosote bush) association, (ii) the preference for saline soils, (iii) isolation near rodent burrows, and (iv) animals as possible agents of dispersal. Results showed that a high percentage of the midden soils contained C. immitis, whereas none of the adjacent, nonmidden soils yielded the fungus. Physicochemical analyses revealed that the dark color and alkaline pH of the midden soils were due to past organic contamination. Repeated isolations were made from soils with low to moderate alkalinity. Alkalinity and sandy texture were consistent features of all soils in this study. However, the lack of any reports of nonsandy infested soils possibly indicates that the sandy texture and alkalinity may be factors in the distribution of this fungus. The organic content, soil parent material, and color were not important in the soil ecology. L. tridentata was not significant in the macroflora at the infested sites surveyed. Samples collected without reference to rodent burrows yielded a high percentage of recoveries. Animals, although not the major natural reservoir, cannot be ignored as possible factors in the ecology of C. immitis. Our knowledge of the ecology of Coccidioides immitis is more advanced than for any other respiratory mycotic disease agent (1). However, as Huppert has pointed out, "A major gap in our knowledge of C. immitis is why the fungus should be so limited in its natural distribution" (10). Others have reported on the uneven, yet consistent, occurrence of the fungus in the soils of the Lower Sonoran Life Zone (15,29). At one site, an area of approximately 2.5 m2 has been positive from 1954 through this study, whereas the surrounding soil has yielded only rare positives. The sporadic occurrence of the fungus in nature seems to contradict its laboratory physiology. It grows rapidly on all common media at temperatures of 20 to 30 C and is not exacting in its nutritional requirements. Growth occurs between pH 3.5 and 9.0 and on clay through sandy soils (13). The fungus is known to infect mam-'Present address: Department of Plant Pathology, University of California, Riverside, Calif. 92502. mals, reptiles, fish, and amphibians (30) and thrives on parts of many desert plants (23). Various theories have been advanced to explain the spotty distribution of C. immitis in the soils of endemic regions. Emmons (9) felt that the carcasses, sputa, urine, feces, and purulent materials of infected rodents were the sources of the pathogen in soil. Maddy (14) emphasized the presence of Larrea tridentata (creosote bush) at his isolation sites in Arizona. Egeberg and Ely (6) recovered the fungus from 13.6% of soil samples taken within 5 ft (15.24 m) of animal burrows, whereas only 3.4% of the samples collected further away were positive. Egeberg and his associates (7,8) reported that high soil salinity was related to increased recovery of C. immitis and the suppression of antagonists. Swatek (27) noted that repeated isolations had been made from old Indian campsites and suggested a possible relationship between the fungus and the increased organic content of the soil on these sites. The purpose of these studies was to define the most important ecological components for C. immitis infestation at former Amerindian habitation areas. Studies were undertaken to determine the relationship between the fungus and the midden soil (areas rich in charcoal, obsidian chips, and other evidence of domestic contamination) and the differences between midden and adjacent, nonmidden soil. MATERIALS AND METHODS Soil collection. Soils were collected aseptically from former Amerindian village sites having no known history of human infection in areas endemic for coccidioidomycosis. Villages in the widely separated Kern and Madera counties of California were chosen for this study, along with control midden soils from sites culturally positive for C. immitis from San Diego, Kern, Butte, and Merced counties (see Fig. 1 .). Amerindian habitation sites may be delineated by various criteria. In this study they included: (i) soil darkened by domestic contamination near watersources (some only seasonal) and, (ii) macroscopic artifacts of human occupation such as obsidian flakes or tools; bedrock mortars or metate stones; "housefloor" depressions of semisubterranean homes; soapstone, shell or glass beads; human remains and petroglyphs. Counties in California from which Amerindian, archeological soils originated. C. immitisinfested control soils (four) or sites (two) were from Butte, Kern, Madera, and San Diego counties. Two sites implicated in human infection were sampled in Fresno and Madera counties. Ten random sites with no known coccidioidal histories were chosen in Kern (three) and Madera (seven) counties. Additionally, cooperating archeologists collected soils from four random sites in Madera County. Determination of points for soil sampling within the middens was made by the use of grids or traverses. Collections of adjacent, nonmidden soils were made at the nearest area which resembled the midden in exposure, drainage and foliage. Soil isolation. The technique of soil isolation employed was basically the double-pour, antibioticfortified, yeast extract agar method (28). Modifications included using 10 g of soil in 90 ml of sterile, distilled water in milk dilution bottles and agitating the 1-in-10 soil suspensions for 1 min and again at 20 and 40 min, and then leaving them undisturbed for 20 min. The plates were incubated at ambient room temperature (23 to 27 C) in a humidified chamber for 3 to 5 weeks. The colonies were examined macro-and microscopically for similarities to C. immitis with consideration for the wide morphologic range possible (11). All fungi demonstrating arthroaleuriospores (20) or racquet cells, or both, were subcultured on Sabouraud dextrose agar. If, on examination of the subculture, it was felt there was still a resemblance to the pathogen, intraperitoneal inoculation of mice was carried out with an aqueous spore suspension. Only fungi which produced spherules and endospores in mice were reported as C. immitis. Soil characterization. Selected midden and adjacent soil samples were compared physicochemically. Carefully mixed individual or composite (combined by equal portions) samples were passed through a no. 10 mesh screen. They were subjected to the following analytical scheme (manufacturers' procedures were followed unless noted): (i) color was determined on dry and paste portions by using Munsell soil color charts (Munsell Color Company, Inc.); (ii) textural classification was made by use of stainless-steel, heat-sterilizable soil sieves; (iii) measurements of pH and Eh were made with a Beckman G pH-meter on 1:5 double distilled water, soil extracts, or soil pastes; (iv) electrical conductivities were determined with a model RC-16B2 Industrial Instruments, Inc., conductivity bridge on pastes and 1: 5 extracts; (v) levels of principle ions were delimited with a Simplex soil testing kit (Edwards Laboratory), and (vi) organic and inorganic carbon and organic nitrogen determinations were made by wet combustion and micro-Kjeldahl techniques, respectively, on powdered (to pass 400 mesh), autoclaved soils (2,12). Soil comparisons. Comparisons of positive midden soils were made with analyses of other infested soils reported in the literature (8,19), by personal communications and on soil samples received. The following persons provided either physicochemical data or samples of C. immitis-infested soils: J. L. Converse RESULTS Soil isolation. Ten sites of former Amerindian villages, with no known histories of coccidioidal infection, were chosen to represent the test middens. Two sites (4-Mad-117 and Fre-SFSC-1) associated with past infection, whose soils had never been studied mycologically, were included as additional controls. Table 1 summarizes the histories of infection and verification of prolonged human occupation at the midden sites surveyed. Results of the soil isolation of C. immitis are tabulated in Table 2 Soil characterization. The results of most of the physicochemical analyses of midden and adjacent, nonmidden soils are compiled in Table 3. The levels of principle ions in the 23 midden and adjacent soils may be summarized as follows: Mn2+, less than 1 ppm; K+, 15 to 20 ppm; Ca2+, less than 40 to 150 ppm; Mg2+, less than 2 to 6 ppm; Al3+, less than 3 ppm; Fe3+, less than 2 ppm; NH,+, up to 2 ppm; NO3-, less than 2 to greater than 25 ppm; NO2-, less than 1 to less than 3 ppm; P03-, 0.5 to 5.0 ppm; Cl-, less than 20 ppm; and SO,-, less than 150 to 600 ppm. Soil comparisons. Comparisons of positive midden soils with other C. immitis-infested soils in the literature, by personal communication and analyses, revealed that: (i) sandy-textured soils occurred in 98.0% of 51 samples (conflicting results were discovered for three of four soils studied by both us and Orr [19], and only the results of the mechanical analyses were used), (ii) alkaline soils occurred in 96.7% of 62 soils, (iii) organic carbon values for 12 soils ranged from 0.21 to 1.80% weight, (iv) organic nitrogen values for 21 soils ranged from 0.029 to 0.190% weight, (v) total organics for 47 soils ranged from 0.39 to 3.13% weight, and (vi) electrical conductivity values for 56 soils ranged from 37 to 27,000 x 10-6 mhos/cm at 25 C. C. immitis has been isolated from soils developed from diverse parent materials: granitic, volcanic, and sedimentary rocks (either stream or ocean derived) and alluvium or lacustrine deposits. DISCUSSION It is evident that a single factor does not determine the distribution of C. immitis in the soil of the Lower Sonoran Life Zone. Emmons has refuted his original hypothesis that rodents are the major reservoir of the pathogen in nature (1) and this is supported by the reports of Swatek et al. (29). Yet, Sorensen (24) has indicated that spherules and endospores protected by body fluids might survive long enough in the soil to allow mycelial growth. Maddy and Crecelius (16) reported the establishment of the fungus in soil with infected animal tissues. During the present study it was noticed that animal activity was increased on the midden sites (221 burrows were counted on eight 188-M2 plots at six different middens compared to 146 on an equal number of adjacent plots). Animals, although not the major reservoir, cannot be ignored as possible factors in C. immitis dispersal. Correlation of the distribution of C. immitis and the macroflora of the positive sites was unproductive. The sites ranged from sparsely vegetated deserts through oak woodlands with scattered pine (Fig. 2). Creosote bush was observed near only 1 positive site (IK-2) out of the 11 included in this survey. The repeated isolation of C. immitis from areas having vegetation markedly different than that of L. tridentata regions should broaden the search for C. immitis-macrofloral associations in nature. Riker (23), in her report on the growth of the pathogen on parts of desert plants, described the inhibition caused by creosote bush. She also noted that the parts of several plants, among them six Opuntia spp. (prickly pear cactus), supported abundant growth and sporulation of the fungus. Campins (5) and Mayorga (18) observed the same genus in endemic areas of Guatemala, Honduras, and Venezuela. The genus is also common in endemic regions of the United States and Mexico. It is possible that the fungus does form alliances with the macroflora, but they may be casual rather than distribution affecting. Egeberg and Ely (6) published a report that incriminated the soil near rodent burrows as a source of C. immitis in nature. Midden samples taken without reference to animal burrows in this research yielded 9.5% positives (of 325 samples), which would indicate that other factors may also be important. Elconin et al. (8) found a positive correlation between the recovery of C. immitis and high soil salinity. Repeated isolations (32 positive samples) from sites in this study revealed markedly less salinity (114 to 1,856 x 10-6 mhos/cm at 25 C) and may indicate that the high salinity might have been a local phenomenon, which, rather than limiting its distribution, represents a more halotolerant extension of the fungus physiology. Swatek's contention (27) that C. immitis infestation is enhanced in the soils of former Amerindian villages has been borne out by this study. However, it appears that sandy texture and alkalinity were more important than the organic content of the soil in this relationship. The pathogen was recovered from 8.1% of 395 soils cultured. Considering just the midden soils, 9.8% were positive, or 8.7% after subtracting the control soils. Soils of 5 of the 10 random midden sites contained the pathogen. Additionally, 1 of 4 random sites, from which soils were collected by cooperating archeologists, was positive. Mention should also be made that the Santee site (29) was another positive midden with no prior history of C. immitis isolation. One of the two sites suspected of causing human infection, 4-Mad-117, yielded the fungus. Historically, random soil samples from en-demic areas have yielded between 2.0 and 3.4% positives for C. immitis (6,9,27). Considering these figures, the percentage of positives reported in this study would appear to be significant. The lack of any positives among the nonmidden soils supports this contention. The epidemiological impact of the association of C. immitis and midden soils will be presented elsewhere. Darkening of the soil was a very stable character of the midden sites and was undoubtedly influenced by past human habitation. During cyclic periods of occupation, the soil was the final receptacle of charcoal, wood, thatch, domestic scraps, human wastes, and burials. Over as much as 1,500 years (at CaMad-173 and the Inyokern Cave), this amounted to a considerable localized increase in organic contamination. Soil color may be related to drainage, aeration, chemical content, and climate. In the midden soils, which were sandy, well drained, low in soluble iron and manganese, and situated in temperate, semiarid to arid regions, the color must be attributed directly to organic materials and charcoal. The soil was alkaline at the middens surveyed. In general, except for IK-3 (which was in an alkaline area), the pH of the surrounding soil was lower. This was due to the accumulation of ash minerals and organic debris. Soil alkalinity is directly related to the intensity and duration of human occupation as revealed in a study of a Chowchilla River Valley site where a proportional increase among pH, artifact yield, and depth of the midden was found (17). Preliminary studies (by D. Rosenberg and J. Kelly, Department of Anthropology, California State University, Long Beach) indicated a similar pattern at CaMad-173. Assessment of organic contamination at contemporary sites of human activity (since 1850) was not part of this study, but it was a contributing factor at two of the sites investigated. CaMad-173 had hydraulic gold mining and ranching activities until the present, and IK-2 had the foundations of a small building on it. Campers, sheepherders, and transients have been observed on some of the sites during this research. All the positive soils studied exhibited an Eh range of 88 to 266 +mV (uncorrected). The Eh-pH milieu occupied by C. immitis in nature appears to be naturally segregated when compared by the method of Baas-Becking (3) with adjacent, nonmidden and random Southern California soils (Fig. 3). Midden soils tended to be higher in carbonates (0.05 to 1.55%) and phosphates (1.0 to 5.0 (29); however, the samples included here did not yield the pathogen. ppm) than adjacent soils (0.02% and 0.5 to 1.8 ppm, respectively). Except for sites liable to seasonal flooding (Fre-SFSC-1, IK-2, and IK-3), all middens demonstrated strong to violent effervescence when tested with 10% hydrochloric acid. Bone and shell, rich in these ions, were part of the aboriginal contamination of the middens. These components may have ecological significance as buffering agents. Comparisons of infested soils revealed physicochemical characters similar to those reported by Cameron in studies of California desert soils (4). Possibly, ecologically limiting factors for C. immitis may include soil pH and texture, whereas nonlimiting factors would include color, organic content, salinity, and soil parent materials. Stotzky (25,26) reported that a positive correlation existed between the presence of clay minerals and the isolation of Histoplasma capsulatum from soil, but no conclusions could be formed concerning C. immitis. Analyses of soils collected for this study revealed that some contained montmorillonite, but the correlation with the presence of the pathogen was not as good as obtained with H. capsulatum (Stotzky, personal communication). It is evident that C. immitis is a physiologically versatile organism, yet the contradiction remains that is has a spotty distribution in nature to the limit of present soil isolation FIG. 2. The macroflora of sites positive for C. immitis ranged from sparsely vegetated deserts (Inyokern Cave, above) to oak woodlands with scattered pine (4-Mad-118, below). techniques. The limiting factor may be the competitive saprophytic ability of the fungus. Observations of its behavior in the mixed cultures of soil isolation plates and comments of other workers support the hypothesis that C. immitis may be a poor competitor for nutrients and biological space. Another consideration is that the consistent soil physicochemical characters determined in this study may well be those most favorable for control of its microbial competitors. Manipulation of these conditions should be considered for the control of the saprophytic phase of C. immitis. The results of this research indicated that C. immitis is strongly associated with Amerindian middens in California. Comparisons of infested soils in the literature and in the laboratory revealed that the chief factors for this association were the presence of alkaline and sandy soils. At the midden sites surveyed, the soil alkalinity and color were due to past accumulation of domestic contaminants.

v3-fos

2018-12-05T10:43:47.708Z

1974-01-01T00:00:00.000Z

56055815

s2

Evaluation of Winter Durum Wheat for Kansas Durum wheat (Triticum d u r u m L.) is an important crop for making pasta, bulgur, couscous, and other products. Annual productions are approximately 100 million bushels in the U.S. and 1200 million bushels in the world. Nearly all of this is spring durum, which is planted mostly at the end of winter and harvested in the summer, but some is planted in the autumn in areas where the climate is mild. Yields of durum compare favorably with yields of bread wheat in the U.S. During the past 10 years, yields averaged approximately 37 bu/a for durum, 33 bu/a for spring wheat, and 39 bu/a for winter wheat. In addition, growers often receive a higher price for durum wheat than for other classes. From 1989 to 1998, durum sold for an average of about $5.00/bu, hard red spring wheat for $4.30/bu, and hard red winter wheat for $4.00/bu. Many of the agronomic characteristics of durum wheat are inferior to those of the hard red winter (HRW) wheat grown in Kansas. Yields of spring durum varieties are often lower than those of HRW wheats, which become established in autumn, rapidly resume growth in the spring, and ripen before the onsets of drought and high temperatures in the summer. The few experimental lines of winter durum that have been available for evaluation often lacked adequate cold hardiness to survive Kansas winters and also matured later than HRW wheat varieties. However, durum wheat is reportedly more tolerant to drought than HRW wheat and may have an advantage in areas where precipitation is low. Little winter durum wheat is grown anywhere in the world, but the crop might have some potential in Kansas. Of 50 experimental lines of winter durum wheat evaluated, many were resistant to leaf rust and lodging, had desirable agronomic traits, and produced high yields of grain. -For winter durum wheat to become a successful crop in Kansas, improvement is needed in winter hardiness, earlier maturity, and quality of the grain for pasta and other products. If a program is undertaken to develop winter durum as a crop for the state, many years will be needed to combine all the attributes into improved varieties and determine the optimum agronomic practices for production. *Graduate student, former professor of agronomy, and professors of agronomy, respectively. Contribution no. 00-172-S from the Kansas Agricultural Experimental Station. Contents of this publication may be freely reproduced for educational purpose. All other rights reserved. In each case, give credit to the author(s), name of work, Kansas State University, and the date the work was published. Kansas State University Agricultural Experiment Station and Cooperative Extension Service, Manhattan, Kansas 66506 EVALUATION OF WINTER DURUM WHEAT FOR KANSAS Emin Donmez, Rollin G. Sears, James P. Shroyer, and Gary M. Paulsen* Durum wheat (Triticum d u r u m L.) is an important crop for making pasta, bulgur, couscous, and other products. Annual productions are approximately 100 million bushels in the U.S. and 1200 million bushels in the world. Nearly all of this is spring durum, which is planted mostly at the end of winter and harvested in the summer, but some is planted in the autumn in areas where the climate is mild. Yields of durum compare favorably with yields of bread wheat in the U.S. During the past 10 years, yields averaged approximately 37 bu/a for durum, 33 bu/a for spring wheat, and 39 bu/a for winter wheat. In addition, growers often receive a higher price for durum wheat than for other classes. From 1989 to 1998, durum sold for an average of about $5.00/bu, hard red spring wheat for $4.30/bu, and hard red winter wheat for $4.00/bu. Many of the agronomic characteristics of durum wheat are inferior to those of the hard red winter (HRW) wheat grown in Kansas. Yields of spring durum varieties are often lower than those of HRW wheats, which become established in autumn, rapidly resume growth in the spring, and ripen before the onsets of drought and high temperatures in the summer. The few experimental lines of winter durum that have been available for evaluation often lacked adequate cold hardiness to survive Kansas winters and also matured later than HRW wheat varieties. However, durum wheat is reportedly more tolerant to drought than HRW wheat and may have an advantage in areas where precipitation is low. Kansas State University Agricultural Experiment Station and Cooperative Extension Service The availability of advanced experimental lines and improved varieties of winter durum from wheat research programs in the U.S. and overseas, the several advantages of durum wheat, and the need to diversify agriculture prompted a reassessment of the crop's adaptation to Kansas conditions. The objective of this study was to evaluate grain yield and other agronomic characteristics of improved winter durum lines and varieties and compare them with popular varieties of HRW wheat in the state. Procedures Sixteen advanced winter durum lines from Oregon State University, 10 lines from the CIMMYT wheat program in Turkey, 7 varieties from Romania, 10 varieties from Hungary, and 7 varieties from the Ukraine were evaluated at Hutchinson and Manhattan during the 1998-99 season. Five HRW wheat varieties (Karl 92, Ike, Jagger, TAM 107, and 2137) were grown in separate experiments at the same locations for comparisons. The soil at the South Central Experiment Field at Hutchinson was Clark-Ost complex, 0 to 1% slope, fine loamy mixed thermic, typic Calciustalls, and that at the North Agronomy Research Farm at Manhattan was Reading silt loam, 0 to 1% slope, fine mixed mesic, typic Arqiudalls. Fertilizer was applied before planting to provide 70 lbs/a N and 27 lbs/a P at Hutchinson and 90 lbs/a N and 27 lbs/a P at Manhattan. Wheat was seeded at the rate of 90 Ibs/a at both locations. Entries were planted in 5-ft-long, singlerow plots on 21 October 1998 at Hutchinson and on 25 October 1998 at Manhattan. The experiments were arranged in randomized complete block designs with two replications at both locations. Production practices recommended for HRW wheat were used for both classes. Glean herbicide was applied at the rate of 0.35 oz/a on 26 January 1999 at Hutchinson and on 2 February 1999 at Manhattan. Observations were made on seedling emergence during the autumn of 1998 and on plant survival, heading and maturation dates, lodging and leaf rust reactions, height and spike lengths, grain and biomass yields, and grain yield components during the spring of 1999. Leaf rust infection was rated on a scale of 1 (no rust) to 9 (complete infection). Plant height was measured from the soil surface to the top of the main spikes at maturity. Spike number, spike length, and spikelets per spike were determined 1 week before harvest. All entries were harvested on 5 July 1999 at Hutchinson and on 1 July 1999 at Manhattan. Plants were cut near the soil surface, dried at 122 o F for 72 hours, and weighed for total biomass. The grain was threshed with a plot thresher and weighed, and yields were adjusted to 12% moisture content. Harvest index was calculated as the ratio of grain yield to total biomass of the dried samples. Kernel weight was measured by counting and weighing 1000 kernels of each sample. Weather conditions were generally favorable for production of winter wheat during the 1998-99 season. Temperatures were above the long-term means at both locations, particularly during the winter. Precipitation from September through June was approximately 4.7 in. above the mean at Hutchinson and 15.7 in. above the mean at Manhattan. Much of the excess precipitation occurred during the critical jointing through maturation stages of wheat from April through June. Results All 50 durum lines and varieties and the five HRW wheat varieties emerged and formed full stands during autumn (data not shown). However, during winter, several durum lines were injured at Hutchinson, and one durum line was killed and five durum lines were injured at Manhattan. None of the HRW wheats was injured at either location. The durum wheats headed over a 5-to 6-day period, and the HRW wheats over a 3-to 4-day period. Although the durum lines headed about 7 to 10 days later than the HRW wheats, they matured only 3 to 4 days later, so their grain-filling duration was nearly 1 week shorter. Leaf rust on durum ranged from none to complete or nearly complete infection at Hutchinson (Table 1) and Manhattan (Table 2). Infection on the HRW wheats, in contrast, ranged from moderate to severe at Hutchinson and mild to moderate at Manhattan. Neither the durum wheats nor the HRW wheats lodged significantly at either location (data not shown). The durum wheats were considerably shorter than the HRW wheats at both locations (Tables 1 and 2). At Hutchinson, all the durum wheats were shorter than the HRW wheats, whereas some overlapping of heights of the two classes occurred at Manhattan. On the other hand, spike length was greater for all the durums than for all the HRW wheats at both locations. Spike density was low for some durums, because winter injury reduced the stands (Tables 1 and 2). All the HRW wheats had excellent stands at maturity and produced over twice as many spikes as the durum wheats at Manhattan. However, all durums formed more spikelets than the HRW wheats at both locations, probably because they had longer spikes. Total plant biomass was high for durum wheats at Hutchinson, except for lines that were injured by cold (Table 1). At Manhattan, where injury was more severe, both the range and mean amount of plant biomass were low ( Table 2). The HRW wheats produced slightly less biomass at Hutchinson but nearly twice as much biomass at Manhattan compared to the durum wheats. Grain yield of the durum wheats ranged widely at both locations, reflecting differences in winter injury, maturity, and other traits of the lines (Tables 1 and 2). Mean grain yield of all the durum lines equaled that of the HRW wheat varieties at Hutchinson but was only about one-half of the mean HRW wheat yield at Manhattan. At both locations, however, the highest durum yields equaled or exceeded the highest HRW wheat yields. Harvest indices of the durum lines reflected the variation in grain yields. The mean harvest index of the durum wheats was low, but some lines had high values at both locations. Harvest indices of the HRW wheats were usually high and varied over a small range. Kernel weights of most durum lines were high at Hutchinson (Table 1) but were low at Manhattan (Table 2). However, some lines had heavy kernels at both locations, which was unexpected because of their late maturity. Kernel weights of the HRW wheats varied over a small range and differed only slightly between the two locations. A large number of durum lines had promising attributes for important agronomic traits at Hutchinson (Table 1). Spike density and harvest index were the only desirable traits that were low in many of the lines. At Manhattan, spike density, kernel weight, and grain yield were frequently unfavorable (Table 2). At both locations, however, some durum lines had all the components for excellent grain yields: high spike density, numerous spikelets per spike, and heavy kernels. Discussion Winter durum wheats exhibited several defects that must be corrected before the crop can be successful in Kansas. However, some of the lines performed well, particularly at Hutchinson, giving promise that production of durum wheat in the state is feasible. A concerted effort to eliminate the undesirable traits and combine the desirable traits into single varieties by breeding could result in a crop that is as well adapted to Kansas as HRW wheat. The most important defects in the durum lines tested were their susceptibility to winter injury and late maturity. The susceptibility to winter injury is disturbing, because it occurred in a year that was mild at both Manhattan and Hutchinson. Normal low temperatures undoubtedly would have caused considerably more injuy. However, the wide range in winter survival among the lines indicated that considerable genetic variability exists in winter durum to improve its cold hardiness by breeding. Late maturity is undesirable in wheat because hot, dy conditions during the last days of June shorten the grain-filling period, shrivel the kernels, lower the test weight, and reduce the grain yield. Unfortunately, the winter durum lines were uniformly late and had little genetic variability for maturity, so improving the maturity by breeding would be slow and difficult. The high kernel weight of some of the lines probably resulted from their superior resistance to drought and heat. Because of this excellent resistance to environmental stress, winter durum varieties might not have to mature as early as adapted HRW wheat varieties in order to be productive. Many of the other traits of the winter durum lines were favorable for production of the crop in Kansas. The plant height and resistance to lodging indicated that winter durum can be grown at high plant density and with high rates of nitrogen fertilizer where moisture is available. The long spikes with numerous spikelets and large kernels suggested that the yield potential is high. These traits probably resulted in the good yields that were obtained from lines that had not been selected for adaptation to Kansas conditions. The high amount of total biomass indicated that winter durum might be used for pasture as well as for grain production. The low incidence of leaf rust in some of the lines suggested that they were resistant to present races of one of the most important diseases of wheat in Kansas. Numerous traits that were not considered in the present study must be evaluated before winter durum wheat can be recommended for production in Kansas. Resistance to the many diseases besides leaf rust that affect wheat, resistance to common insects, and quality of the grain for making pasta and other products must be determined. If durum wheat appears to be feasible for Kansas, several decades may be needed to combine all the essential traits into improved varieties for production by the state's growers. Additional research will be needed to determine the crop's agronomic requirements, such as planting date, seeding rate, and fertilizer rate, and the optimum areas of the state for production. Winter durum wheat may be a promising crop, but fulfillment of its promise is many years away.

v3-fos

2020-12-10T09:04:12.602Z

1974-01-01T00:00:00.000Z

237233570

s2

Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion. Diplodia maydis (Schw.) Lev is one of the major pathogens causing stalk rot (4) of corn (Zea mays L), a complex disease resulting in an annual loss of more than 109 bushels of corn in the corn-growing areas of the world (8). Many studies concerning resistance or susceptibility of corn to D. maydis have been reported (see 4,8), but only a few studies have been reported on the in vitro growth of D. maydis (1,2,6,10,11), and none of these at the ultrastructure level. The paucity of information concerning the gross morphology of D. maydis led us to examine (via scanning electron microscopy) the ultrastructural morphology of D. maydis grown on laboratory media and natural substrates to lay ' Present address: Center for Electron Microscopy, Southern Illinois University, Carbondale, Ill. 62901. 'Present address: University of Delaware, 104 Hullihen Hall, Newark, Del. 19711. the groundwork for further studies of host-parasite interaction. Growth conditions. Cultures were maintained at 25 C on sterile oats inoculated with D. maydis and on potato dextrose agar plates. The inoculum for the liquid media and PDA plates was prepared by transferring a 5-mm plug of D. maydis grown for 4 days on PDA. Oats were inoculated with a few infected oat kernels from maintenance cultures of D. maydis. Spores were obtained from maintenance oat cultures by adding 10 ml of sterile distilled water into an inoculated oat flask, allowing spores to swell for 10 to 15 min with occasional gentle agitation of the flask, and decanting the suspended spores. For purposes of comparison, D. maydis was grown at 25 C in MR2 medium on a rotary shaker, a reciprocal shaker, and under stationary conditions. In other studies D. maydis was grown for 5 days at 25 C in CPK, MR1, or MF2 media. Corn kernels and corn husk tissue from field-grown plants infected with D. maydis were provided by A. L. Hooker. Fixation and specimen preparation methods for scanning electron microscopy. To determine a suitable fixation procedure for D. maydis mycelia and spores, the glutaraldehyde and Parducz fixation methods were tested. In glutaraldehyde fixation, mycelia and spores were immersed for 1 h in 0.1% s-collidine-buffered glutaraldehyde (Electron Microscopy Sciences), followed by three washes in buffered s-collidine and a subsequent 1-h fixation in 1% s-collidine-buffered glutaraldehyde, followed by 10 washes in s-collidine buffer. In Parducz fixation (9), mycelia and spores were immersed for 10 min in 6 parts of 2% OsO4 to 1 part of saturated HgCl2, followed by 10 washes in a buffered solution. Both fixatives were used alone as well as in conjunction with each other. Freeze-dried (Edwards Pearse tissue dryer) preparations were compared with air-dried preparations on all samples examined. Mycelia grown on PDA were compared by using freeze-drying, critical-point drying (Bomar 900EX or Denton criticalpoint drying apparatus), and air-drying. Scanning electron microscopy. Fixed and dried D. maydis mycelia, infected corn kernels, corn tissue, and infected oat kernels were attached to Cambridge specimen stubs by using double sticky tape slightly melted with acetone. Spores were pipetted onto specimen stubs by the method of Murphy and Campbell (7). Samples were rendered electrically conducting by evaporating a thin 40:60 palladium gold alloy coating onto the surface in a vacuum evaporator while the samples were simultaneously tilted and rotated to insure even coating. The specimens were examined in a Cambridge Stereoscan Mark 11A scanning electron microscope operated at 20 kV. RESULTS Fixation results. Glutaraldehyde fixation followed by Parducz fixation and freeze-drying gave the same morphological preservation as Fig. ld, illustrating well-preserved spores). Glutaraldehyde fixation followed by freeze-drying resulted in mycelia and spore collapse. All air-dried samples and the samples unfixed and freeze-dried resulted in collapse of mycelia and spores as well as a matting of the mycelia and slime material. Freeze-drying versus critical-point drying. In comparing preparation methods, mycelia were preserved equally well with fixation and freeze-drying ( Fig. 2a and b) and fixation and critical-point drying ( Fig. 2c and d). The advantages and disadvantages of each method are discussed by Boyde and Vesely (3) but, considering equal preservation, critical-point drying is of greater advantage because the drying time (45 min) is considerably less than that for freeze-drying (4 h). Rotary versus reciprocal shaker cultures versus stationary cultures. D. maydis mycelia obtained from rotary shaker and from reciprocal shaker cultures were well preserved and had a similar overall appearance ( Fig. 3a and b). D. maydis grown in stationary cultures, however, was found to be surrounded by slime-like material. The copious precipitate on the mycelia surfaces is illustrated in Fig. 4a and b, and a coalescence of the slime material is shown in Fig. 4c. Coalescence of slime-like material does not appear to be an artifact of fixation since unfixed controls showed the same type of coalescence. Comparisons on different growth media. (i) MR2 medium. Figure 3a illustrates the mycelial organization in a mycelial pellet. Fixed and air-dried samples collapsed, and mycelia appeared to be matted. This is even more evident in samples which were air-dried without fixation. Figure 4a illustrates the overall mycelial appearance after growth for 4 days in stationary cultures. Copious particulate matter is found on the surface of mycelia grown in stationary cultures (Fig. 4b); also apparent is the coalescence of slime-like material over the mycelia, as indicated by an arrow in Fig. 4c. This might be expected, as copious quantities of slime-like material are elaborated during the growth of D. maydis in liquid media (1,2,6). The greatest amount of the precipitate is found on the mycelia grown under stationary conditions. Figure 5 shows a finely laced net randomly appearing throughout the mycelia with small bead-like structures interspersed along the net. The amount of netting interlaced in the mycelia was well correlated with the amount of slimelike material elaborated by the fungus during growth. The fungus grown in MR2 medium elaborated the least amount of slime-like material when compared with growth in MR1, MF2, and CPK media. (ii) MF2 medium. The mycelial pellet has a slightly different overall appearance when grown in MF2 medium in that it showed less regularity than found in MR2 medium. Typically, a film of secreted material overlying and stretched between the mycelia is apparent (Fig. 6a). Compared with cultures grown in MR2 medium, there is a greater abundance of beadlike structures interlaced throughout the mycelia (Fig. 6b and c), and larger thickened node-like structures are observed. The mycelia from cultures grown in MF2 and MR2 media both have a smooth surface with no discernible characteristic structures. (iii) CPK medium. When grown in cpk medium, the mycelial pellet has a honeycomblike appearance. In juxtaposition, one can see the honeycomb netting and smooth-surfaced mycelia (Fig. 7a); in certain areas, the honeycomb-like appearance takes on a more or less regular pattern (Fig. 7b). Mycelia were of two types: those covered with particulate material and those with a smooth surface, relatively free of other material. The bead-like structures and the coalescing of these structures noted in cultures grown in MR2 and MF2 media are also apparent in cultures grown in CPK medium ( Fig. 8a and b). The thickened node-like structures observed in MF2-grown cultures were not observed in CPKgrown cultures. (iv) MR1 medium. After 6 weeks of growth under stationary conditions in MR1 medium, the mycelial pellets were engulfed in a gel-like matrix; sporulation had occurred and the formation of bead-like structures and a coalescence of slime material were also noted. The mycelium appeared to be composed of a continuous series of bulges ( Fig. 9) suggestive of intercalary spores, although these are not reported to occur in this organism. (v) PDA medium. After 5 days of growth on PDA plates, coalescing of slime material on the smooth-surfaced mycelium, thickened nodes, and bead-like structures was observed. However, nodes and bead-like structures were not as abundant as in cultures grown in liquid media. This may be due to the relatively dry growth conditions in agar plate culture. D. maydis spores are ovate, most commonly two-celled and slightly curved with rounded to tapered ends. Figure 10a illustrates the most commonly observed spore shape where a septum between the cells can be noted. Air-drying of D. maydis spores mimics the condition in which the spores are found in naturally infected tissues. Air-dried spores are generally flask shaped (Fig. 10c) or collapsed (Fig. 10b). The average size of noncollapsed spores was 25 by 4 ,um. Size calculations are only approximate due to the variable specimen to beam angles inherent in the scanning electron microscope. (vi) D. maydis on oat substrate. On oats, D. maydis produces thousands of spores in small, black, flask-shaped pycnidia (Fig. 11a). Although the pycnidia may be globose, flask shaped, or irregular in shape, a typical pycnidium is illustrated in Fig. lib and c. Pycnidia are filled with spores (Fig. lie), and an opening (Fig. 11d) in the pycnidium for the dissemina-tion of spores can be seen. Inside the pycnidium, a fine-laced netting is found interspersed between the spores (Fig. 12a). A few of the bead-like structures noted in cultures grown in liquid media are also present (Fig. 12a). The interior of some of the pycnidia appears filled with spores engulfed in a thick gel-like matrix (Fig. 12b). Possibly the laced netting thickens to a gel-like matrix, giving some advantage in preservation under dry conditions. Interpycnidial mycelia are illustrated in Fig. 12c. A thin film overlying and stretching between the mycelia was observed (Fig. 12d), as well as a more extensive coalescing of slime material (Fig. 12e). Node-like structures were also observed in D. maydis-infected oats (Fig. 12f). Mycelia exhibited a smooth surface, and spores were two celled, rounded to tapered, and many were flask shaped due to natural collapse upon drying. Although samples were somewhat naturally dried during normal growth conditions, fixation was found to be necessary, as samples that were not fixed were found to have an artifactual cracked appearance both in the pycnidium and on spore surfaces. (vii) D. maydis on corn husks. The appearance of D. maydis on corn husks was found to be similar to that grown on oats. Small pycnidium dotted the surface ( Fig. 13a and b). Although often random in distribution (Fig. 13a), pycnidia sometimes appear to be closely associated with vascular bundles (Fig. 13b). Pycnidia were filled typically with two-celled, flask-shaped spores (Fig. 13c). The mycelial surface was smooth (Fig. 13d), although it was collapsed because of the naturally dry growth condition. Coalescing of slime-like material was noted interspersed between the mycelia. Less slimelike material covered the spores on the pycnidial surface compared to that found in infected oat tissue. Neither thickened nodes nor bead-like structures were noted in the fungus obtained from infected corn husks. (viii) D. maydis on corn kernels. D. maydisinfected corn kernels were examined from various locations in Illinois (Carbondale, Cornell, Morris, Lincoln, and Varne). Figure 14a illustrates a typical overall view of D. maydis-infected kernel. Pycnidia were not as compact (Fig. 14b) as that found on oats or corn husks. Thickened nodal areas were found in all samples examined. Many of the nodes tended to be round rather than ovate (Fig. 14c); however, the latter-shaped nodes were also present ( 14d), as indicated by an arrow. Mycelia were smooth surfaced without evidence of precipitated material on the surface. Spores were collapsed due to natural drying conditions during growth. DISCUSSION Glutaraldehyde fixation followed by Pardupz fixation and critical-point or freeze-drying resulted in the same morphological preservation as Parducz fixation alone followed by criticalpoint or freeze-drying. Mycelia and spores collapsed if treated with glutaraldehyde fixation followed by freeze-drying or air-drying, or prepared by freeze-drying or air-drying without fixation. From these results, we decided to routinely use the double-fixation procedure for field studies (glutaraldehyde fixation in the field followed by Parducz fixation and criticalpoint or freeze-drying after returning to the laboratory) and eliminating the glutaraldehyde fixation when sampling D. maydis from laboratory cultures. The collapse in mycelia, spores, and pycnidia observed in the air-dried field collections (husks and kernels of corn) are believed to be real and not induced by a fixation procedure. Major variations in fungal morphology were not observed due to culture conditions (rotary, reciprocal, or stationary liquid culture) or media, although the greatest amount of precipitate was observed on mycelia from stationary liquid culture. Apparently, the slime-like material is dispersed by movement in rotary and reciprocal shaker culture. Fine-lace netting with beads was found in all synthetic cultures. Minor differences appeared in the amounts of slimelike material observed and in the presence or absence of thickened nodes. Also apparent in MR1 medium was cell-like bulging of the mycelia. These cell-like bulges are suggestive of intercalary spores; however, these are not reported to occur in this organism, and no crosswalls (indicative of intercalary spore formation) are found in thin-section studies (Murphy, unpublished data). From this study, it appears that the degree of coalescence of slime-like material progresses as follows: the formation of bead-like structures (Fig. 6b); progressive accumulation of these precipitate on mycelia at a higher magnification. Marker represents I um. x5,000. (c) Note coalescence of slime material. Marker represents 10 gm. x 180. All cultures were grown in MR2 medium at 25 C. into a film overlying and stretched between mycelia. This observation compares with that found in MF2-grown cultures (Fig. 6a). Marker represents 10 m. x980. FIG. 9. D. maydis grown in MR1 medium, 6-week stationary culture, Parducz fixed and freeze-dried. The mycelium appears to be composed of a continuous series of bulges. Marker represents I 'sm. x5.100. structures ( Fig. 8a and b); formation of thickened film of secreted material overlying and stretched between the mycelia (Fig. 6a); and finally a coalescence of the slime-like material (Fig. 4a). We believe that the same type of progression results in matting and coalescence of mycelia in the pycnidium. It should be realized that these suggestions are made from studying hundreds of micrographs, and the coalescence sequence is only briefly represented in the five micrographs listed. The slime-like material appears to be different from the material precipitated on the mycelia. However, it may be that the two arise from the same metabolic pathways since both seem to occur simultaneously. Why the great variation in amounts of slimelike material observed with the culture media tested is not known. It may be that the slimelike substance produced by D. maydis plays a role in the prevention of desiccation under normal conditions. One variation that did occur in the mycelial organization in the cultures was the formation of a honeycomb netting of mycelia in CPK medium. Why thickened nodes were observed in MF2 and PDA media and were not found in MR2, CPK, and MR1 media is also not known. Again, in thin-section studies (Murphy, unpublished data), cross-walls are not found. Perhaps the nodes represent storage material in the hyphae and occur only when certain metabolic products are in excess, depending on the food source. The amount of slime secreted by the fungus does not seem to be correlated with the presence or absence of thickened nodes. When comparing morphology of D. maydis on artificial versus natural substrates, the biggest difference appears to be in the formation of pycnidia. On natural substrates, these occurred along vascular bundles of the corn husk and along the oat and corn kernels. The tightness of the pycnidium was least on corn kernels and greatest on infected oats. The presence of finelace netting with beads on infected oats but not on corn kernels or husks, and the lack of thickened nodes in fungal samples from infected corn husks but not corn kernels or oats may indicate significant nutritional and environmental differences resulting in physiological differences in spores of the pathogen. Similarly, there may be differences in these characteristics when host-pathogen responses with a wide variety of cultivars and pathogen isolates are studied. Regardless of these possibilities, from our study we suggest that there should be little morphological differences expected in the pathogen mycelia, spores, and pycnidia. Although scolecospores have been reported (5) Marker represents 1 Aim. x2,300. strains of Diplodia, and including D. maydis, none were observed in the material studied. From this study, we have enough morphological data about this pathogen to identify it if observed in corn stalk tissue infected with D. maydis. With the scanning electron microscopy preparation methods now available, and with knowledge about the morphological differences in various synthetic and natural environments of the pathogen, several other investigations of D. maydis can be undertaken. Of particular interest now is the documentation and clarification of the stalk rot process as D. maydis penetrates its natural host and spreads from cell to cell within the stalk, over kernels, and through the husk tissue. Included in this is the need to study the digestion process and changes in morphology when production of cellulolytic enzymatic enzymes are stimulated and their release occurs (1,2).

v3-fos

2018-04-03T01:55:19.277Z

1974-03-01T00:00:00.000Z

31505090

s2

Interaction of pH and NaCl on enumeration of heat-stressed Staphylococcus aureus. The effect of pH level and NaCl concentrations, alone and in combination, on the enumeration of unstressed and heat-stressed cells of three strains of Staphylococcus aureus was determined. A definite narrowing of the optimum pH range for enumeration of both unstressed and heat-stressed cells was observed as the NaCl concentration was increased from 0.0 to 7.5%. Counts of unstressed cells diminished only slightly with increases in NaCl, whereas heat-stressed cells showed a marked sensitivity to NaCl concentrations of 4% and above, regardless of the pH level. Because of this sensitivity to NaCl, recoveries were far poorer than with unstressed cells at NaCl concentrations of 4% and above. The ability of Staphylococcus aureus to grow at greater NaCl concentrations in comparison to many other bacteria is well documented (10,12). A number of media that use NaCl as the basis of their selectivity have been developed for the quantitative enumeration of this organism (3,4). In some situations, enumeration of S. aureus by such selective media may be difficult if the organism has been subjected to heat treatment or other forms of physical stress sufficient to cause sublethal injury. However, the assumption is often made that conditions satisfactory for the enumeration of unstressed bacteria are equally applicable to heatdamaged bacteria. Busta and Jezeski (2) found that S. aureus cells heated at 60 C for various lengths of time lost their ability to multiply on Staphylococcus medium 110 containing the normal concentration of 7.5% NaCl. Since then other investigators (8,13,15,17) have reported similar observations, indicating a definite NaCl sensitivity by S. aureus after thermal injury. Such a change in NaCl tolerance obviously reduces the effectiveness of media that are designed to selectively enumerate S. aureus on the assumption that the organism is tolerant to high concentrations of NaCl. In addition to a loss of NaCl tolerance after thermal injury, F necessary for the recovery of heat-stressed S. aureus was reduced in comparison to that for the unstressed organism. Other reports on the effects of pH alone or pH in combination with NaCl have been concerned with unstressed S. aureus. For example, Lechowich et al. (11) found that pH 5.6 reduced both aerobic and anaerobic growth of S. aureus in pickling brines used for curing meats, and pH 4.8 prevented growth. landolo et al. (9) demonstrated that the effects of NaCl on unstressed cells were greater either when pH deviated from the optimum or incubation temperature was raised to 45 C. Genigeorgis and Sadler (7) concluded that growth and enterotoxin production were better when initial pH of the medium was increased and salt concentration was decreased. Growth of organisms tended to raise the pH so that the pH at time of enterotoxin production unquestionably was higher than when growth was initiated. Scheusner et al. (14) obtained growth when the initial pH of the broth was 4.96 to 9.02. Genigeorgis et al. (5) reported growth over the pH range from 4.00 to 9.83 in the absence of NaCl, but increasing concentrations of NaCl narrowed the range of both acid and alkaline values, the limits being pH values of 4.50 and 8.0 at 12% NaCl. Nelson (Bacteriol. Proc., p. 20, 1971) reported that the inhibitory effects of pH and NaCl were additive when enumerating heat-stressed coliform bacteria, maximum counts being obtained only over a pH range of 6.0 to 7.0 in the presence of 3% NaCl, whereas unstressed organisms were unaffected over a pH range from 5.0 through 9.2 in the absence of NaCL in the enumeration medium. MATERIALS AND METHODS Test organisms. Three strains of coagulase-positive S. aureus were employed. Strain UA-112 was obtained from the Department of Microbiology and Medical Technology at the University of Arizona. Strains S-6(B) and FRI-100, known producers of enterotoxin, were obtained from M. S. Bergdoll of the Food Research Institute of the University of Wisconsin. The organisms were maintained on brain heart infusion agar (Difco) slants held at 5 C prior to use. Cells were grown in 5 ml of nutrient broth (Difco) for 24 h at 37 C, after which 1 loopful was transferred to 50 ml of nutrient broth contained in 125-ml flasks and incubated for 24 h at 37 C. Thermal stressing and enumeration. A 1-ml sample of a 24-h culture was placed into 5 ml of sterile reconstituted milk (110 g of milk solids-not-fat/liter) contained in screw-cap test tubes (16 by 125 mm). A sample serially diluted in phosphate buffer was plated on unmodified nutrient agar (Difco) by the procedure outlined in Standard Methods (1). Cells of UA-112 were plated on nutrient agar containing NaCl concentrations of 0, 2, 3, 3.5, 4, 5, and 7.5%. Counts at each NaCl concentration were made at pH levels of 5.0, 5.5, 6.0, 7.0, 8.0, 8.5, 9.0, and 10.0. Cells of strains S-6(B) and FRI-100 were plated on nutrient agar containing fewer NaCl concentrations accompanied by fewer pH levels to check only those points of greatest importance. NaCl concentrations of 0, 2, 3, 3.5, 4, and 5% were used, along with pH levels of 5.0, 5.5, 7.0, 7.5, and 9.0. Adjustment of pH was made prior to plating by the addition of either 1 N H2S04 or 1 N KOH to the warm NaCl-nutrient agar. After the plating of the unstressed cells, the remaining milk sample was immersed, along with a control milk sample containing a thermometer, in a water bath maintained at 56 ± 0.1 C. Timing of the sample commenced when the temperature reached 56 C in the control tube. Exposure time of the sample was designed so as to reduce the viable population as determined on nutrient agar by 99% and was 7 min for strain UA-112 and 6 min for strains S-6(B) and FRI-100. A high level of kill was selected to maximize the observed effects of stress. After exposure, samples were cooled in ice water and plated on the same series of media on which the unstressed cells had been plated. Plates were incubated at 37 C for 48 h. All platings of strain UA-112 were made in duplicate, and two runs were made for each NaCl-pH combination. Duplicate platings for strains S-6(B) and FRI-100 were used only for counts on the control unmodified nutrient agar. Two trials were run for each NaCl-pH combination. Statistics. Counts of stressed cells on modified media were expressed as a percentage of the count obtained on unmodified nutrient medium, and this percentage then converted to logarithms to the base 10 to give log percent recovery. Recovery values for unheated and heated S. aureus UA-112 were subjected to a completely randomized two-way factorial arrangement (16) by using NaCl and pH as the factors. A similar experimental analysis for unheated and heated cells of strains S-6(B) and FRI-100 involved a completely randomized three-way factorial analysis of variance, with NaCl, pH, and strain constituting the factors. RESULTS Data on recovery of unheated cells of S. aureus strain UA-1 12 at different levels of pH in the presence of increasing concentrations of NaCl are shown in Fig. 1. A gradual narrowing of the optimal pH was shown as the NaCl concentration increased from 0 to 7.5%. At lower NaCl concentrations, maximum recoveries were obtained over a pH range of 5.0 through 9.0. At NaCl concentrations of 3.5 and 4%, recoveries at pH 9.0 were significantly less than maximum, whereas recoveries at 5 and 7.5% were significantly reduced at pH levels of 5.0, 5.5, and 6.0. Maximum recoveries at higher NaCl concentrations were only slightly lower than recoveries at the same pH levels in the absence of NaCl. Data on recovery of heat-stressed S. aureus strain UA-112 at different pH levels in the presence of increasing concentrations of NaCl are shown in Fig. 2. These heat-stressed cells also showed a gradual narrowing of their optimal pH range as the NaCl concentration increased. Recoveries at 4% NaCl showed an overall decrease from counts obtained at 3.5% regardless of the pH level. Recoveries at 5 and 7.5% NaCl were quite similar to those obtained at 4% in that recoveries were only approximately 10% of the optimum. Recoveries of unstressed and heat-stressed cells of strains S-6(B) and FRI-100 at various pH levels in the presence of increasing NaCl concentrations were so similar to those of strain UA-112 that the data are not presented. Analysis of variance for all three unstressed strains of S. aureus revealed significant firstorder interactions between NaCl and pH, indicating that changes in the level of one factor modify the effects of the other factor on the enumeration of unstressed cells. In addition, comparison of unstressed cells of strains S-6(B) and FRI-100 showed no significant differences between these two strains in their responses to NaCl and pH. Heat-stressed cells of all three strains also showed significant first-order interactions between NaCl and pH, indicating an interactive effect of these two factors on enumeration. Furthermore, comparison of strains S-6(B) and FRI-100 to one another revealed two additional first-order interactions. Significant interactions were noted with the pH and strain and also NaCl and strain factors. These interactions indicate that the two strains reacted slightly differently to pH and over all NaCl concentrations and to NaCi over all pH levels. In comparison to unstressed cells, heat-stressed cells showed more variation from strain to strain in their responses to NaCl and pH, even though both groups show a narrowing of their optimum pH range as the NaCl concentration increases. DISCUSSION Although three strains do not constitute a large sample, the indications are that the responses to pH and NaCl demonstrated here are characteristic of the species. Such responses to pH and NaCl indicate an interaction effect by the two factors on recovery of S. aureus. As the degree of adversity, especially pH, was increased, a gradual decline in count occurred; this decline was accentuated by increasing NaCl concentration. Both unstressed and heatstressed cells showed a narrowing of their optimum pH ranges as the NaCl concentration was increased. The degree of the observed effects very probably could be altered appreciably by variations in the degree of stress. These findings were in close agreement with the results obtained by Genigeorgis et al. (5,6) on unstressed cells of S. aureus, indicating a decrease in count of the organism at any pH level as the NaCl concentration increases. However, due to the marked sensitivity of heat-stressed cells to NaCl, overall recoveries at 4% NaCl and above were far poorer, regardless of pH, in comparison to unstressed cells. Maximum counts of unstressed cells of S. aureus can be obtained quite effectively in a medium containing 7.5% NaCl adjusted to a pH range of 7.0 through 8.5. Maximum counts of heat-stressed cells cannot be attained on media containing 5% or more NaCl. The pH of the medium plus sample should not be outside a range of 5.5 through 8.5, even with an NaCl concentration below 4%. Although dropping the NaCl concentration of a selective medium would obtain higher counts of staphylococci that had been subjected to thermal stress, it would do away with much of the selective character of such a medium. Due to the NaCl sensitivity of S. aureus after thermal stress, it would appear that no combination of pH and NaCl would offer promise of improving recovery of such an organism from mixed populations of bacteria. Improvements of media for this purpose must be sought in other directions.

v3-fos

2018-04-03T06:14:49.429Z

1974-01-01T00:00:00.000Z

26061745

s2

Effect of Amendments on the Microbial Utilization of Oil Applied to Soil were monitored over a 308-day period for changes in bacterial and mold numbers. Changes in the chemical composition of the oil applied to the plots was followed by using chromatographic techniques. Application of fertilizer resulted in a stimulation of bacterial numbers and in the rate of utilization of n-alkane components of the saturate fraction. The application of oil-utilizing bacteria, however, resulted in only a slightly accelerated rate of utilization of n-alkane components of chain lengths C20 to C25. The isoprenoids, phytane and pristane, Oil spills, whether on water or soil, do disappear (6), but very little is known about what can be done to accelerate this process. Recent work by Reisfeld et al. (5) and Atlas and Bartha (1) indicated that the disappearance of oil from sea water could be accelerated by the addition of deficient nutrients such as nitrogen or phosphorus, or both. Suggestions have also been made (4,7) for microbial seeding of spills since bacteria and fungi are the only biological species which have the metabolic capability of utilizing petroleum carbon for cell synthesis. There is, however, very little information in the literature evaluating the effect of such treatments on the acceleration of the utilization of oil spilled under natural conditions. Crude oil is essentially a mixture of carbon and hydrogen, and thus spills will result in an imbalance in the carbon-nitrogen ratio at the spill site. For bacteria to grow efficiently, they require about 10 parts carbon to 1 part nitrogen. If the ratio is greater, e.g. 100:1 or 1,000:1, growth of the bacteria and utilization of carbon source(s) will be retarded. In addition to there being a nitrogen deficiency in oil-soaked soil, other nutrients such as phosphorus may become growth-rate limiting. Therefore, in the experiments described in this paper, urea-phosphate, a fertilizer, was added to oil spilled on soil, thus correcting both deficiencies in one application. A survey of soils (unpublished observations) from the northwest area of Canada for the presence of oil-utilizing microorganisms indi-cated that not all soils have an indigenous population capable of utilizing oil. Thus, oil spills were also inoculated with oil-utilizing bacteria with and without a concurrent application of the urea-phosphate amendment. The experimental site chosen was in the Swan Hills area of north central Alberta, which represents a major oil-producing center in this province. The soils are of low fertility, are in a frozen state for approximately 5 to 6 months of the year, and are representative of soil and climatic conditions existing in the production and pipeline transport areas of this province and western Canada. MATERIALS AND METHODS Field sites. The plots in the Swan Hills area were placed on an overgrown, unused airstrip. Four replicate plots of each treatment, i.e., control, control plus oil, plus oil and bacteria, plus oil and fertilizer, plus oil, bacteria, and fertilizer, were placed in a random manner. The composition of the oil used in the spill is presented in Table 1. This crude petroleum was obtained from the Shell Oil Co. and is representative of producing wells in this area. The oil was applied in mid-July 1972 by sprinkling from perforated cans at a rate of 60 liters of crude oil per 9 square meters of soil, and it completely covered the surface of the plots with a thin layer of oil. Fertilizer, urea-phosphate (nitrogen-P20,-potassium, 27:27:0) was applied simultaneously to eight plots at a rate of 60 g of nitrogen per m2 (equivalent to 600 kg of nitrogen per hectare). A mixed culture of bacteria capable of utilizing an oil of similar quality (Norman Wells crude oil; chemical composition in Table 1) was also at this time applied b Used for growing cells which were applied to plots. c NSO, nitrogen-sulfur-oxygen-containing organic compounds. to eight plots, four of which had received a fertilizer treatment. The cells were grown on a rotary shaker (300 rpm, 1-inch [about 2.5 cm] eccentricity) at 25 C in 2-liter Erlenmeyer flasks containing 1 liter of basal salts medium (2) and 1 ml of Norman Wells crude oil as sole carbon source. The cells were recovered by centrifugation after 96 h of growth, washed, and resuspended in tap water at 4 C. This suspension was diluted to a concentration such that application of 6 liters of suspension per plot yielded an application rate of 101 bacterial cells per cm2. The mesophilic bacterial population used for bacterial seeding was composed of the following genera: Flavobacterium and Cytophaga sp. (41%), Pseudomonas sp. (34%), Xanthomonas sp. (10%), Alcaligenes sp. (9%) and Arthrobacter (5%). Soil samples (total weight approximately 500 g) were taken periodically and analyzed for total bacterial and fungal counts. When such samples were obtained from plots which received an oil treatment, the oil was extracted and its chemical composition was determined by chromatographic techniques (2). Thus, this experimental protocol allowed the statistical analysis of the effect of treatments on the microbial population and on the utilization of the applied oil. Microbiological methods. Changes in microbial numbers were monitored by using a spread plate count technique. Plate count agar (Difco) was used for the enumeration of bacteria, and malt extract agar (Difco) adjusted to pH 4.5 was used for molds. Quintuplicate plates of each dilution were incubated at 21 C for 6 days before counting. The bacteria which comprised the mixed population were classified to the generic level on the basis of the following tests: Gram reaction, presence and position of flagella (determined by electron microscopy); oxidation and/or fermentation of sugars with and without acid and/or gas production; catalase and oxidase activity. Isolates which were classified as Pseudomonas were streaked on Pseudomonas F and P agar (Difco) and observed for fluorescein or pyocyanin production, respectively. Chemical methods. The chromatographic techniques used for the analysis of crude oil was as described in our previous paper (2). These techniques resolve crude oil into asphaltene, saturate, aromatic, and the polar nitrogen-sulfur-oxygen-containing organic fractions. The saturate fraction was further resolved by using gas chromatography as reported previously (2). Recovery of oil from soil. Approximately 100 g of a soil sample was extracted four times with 100-ml portions of n-pentane, and the extracts were combined and evaporated to dryness in a fume hood at room temperature. The dry residue was redissolved in 4-to 25-ml portions of benzene, and, after pooling the benzene fraction, non-hydrocarbon material present was allowed to settle out. A portion of the benzenesoluble fraction was evaporated to dryness, weighted, resuspended in n-pentane, and analyzed by liquid and gas chromatographic procedures (2). Statistical analysis. Before statistical analysis, all data were subjected to the Nalimov test (3), which rejected data outside of the confidence limit of 95%. The means and t tests were made between sets, each possessing a minimum of four observations. All statistical analyses were performed on a minimum of four observations. All statistical analyses were performed by an IBM 360 computer. RESULTS The gas-liquid chromatography (GLC) profile of the n-saturate fraction of the oil before and immediately after application to soil is presented in Fig. 1. The mean pH values for these plots 12, 66, and 308 days after treatment ranged from pH 4.8 to 5.9. Statistical analysis of these data indicated that there were no significant differences between plots as a result of amendment application. Changes in the bacterial count observed 12, 66, and 308 days after treatment are presented in Table 2. These data show that there was a statistically significant increase in bacterial numbers within 12 days of the application of oil when fertilizer had been applied to the plot as well. The stimulation of bacterial numbers decreased within 66 days of treatment, and by 308 days the values for those plots that had received fertilizer, with or without bacteria, were similar. Statistical analysis of these results ( Table 2) indicated a consistent significant difference at the 95% confidence level and occasionally at the 99% confidence level when the effects of the fertilizer amendments on bacterial counts were compared with those values obtained from the control oil plots. Changes in the mold counts are presented in Table 3, and statistical analyses indicated that the application of amendments was without effect on this parameter at the 95% confidence level. The chemical composition of the oil recovered from the plots 12 days after treatment is presented in Table 4. Statistical analysis of these data showed that the application of amendments was without effect on the chemical composition of the oil recovered from the plots. The data in Tables 5 and 6 suggest that the oil recovered 66 and 308 days after treatment from plots which received the fertilizer amendment have a reduced n-saturate content. Statistical _ analysis of this reduction in n-saturate content of the recovered oil indicated a significant reduction (at both the 95 and 99% confidence levels) 66 and 308 days after treatment in those plots which had received a fertilizer treatment. A comparison of the n-saturate profiles 12 days after the oil had been applied to the soil (Fig. 2) shows that the application of fertilizer resulted in a slightly accelerated rate of n-saturate utilization. The change in profile is reflected in a utilization of the middle chain-length Df the satu-(i.e., C18 to C29) n-saturate components. The and after data in Fig. 3 show that the fertilizer application resulted in a complete disappearance of the n-saturate components, with the exception of U1s plots the branch-chain isoprenoids, phytane and pris-10/g after a tane, 66 days after treatment. These data also 308 days suggest a stimulatory effect by the application -308 days of the bacteria at this time which is reflected in _ an increased utilization of the C20 to C25 41.7 chain-length n-saturate compounds. These re-60. 1 suits are confirmed in Fig. 4, where a compari- dition can be responsible, in part, for the persistence of oil applied to soil has been substantiated by the data presented in this paper. The application of nitrogen and phosphorus as urea-phosphate resulted not only in a rapid increase in the numbers of bacteria present but also in an accelerated rate of disappearance of the n-saturate fraction of the crude oil applied to the soil. However, the efficacy of the application of oil-utilizing bacteria is still unanswered at this time since the effect reported, i.e., the slightly accelerated utilization of n-saturates of chain lengths C20 to C25, was not observed on all replicates. The low degree of change observed between the 66-and 308-day samples can be accounted for by the fact that for most of this period the area was in a frozen state. The persistence of the isoprenoids, phytane and pristane, in the GLC profile of those plots where n-saturation utilization occurred (i.e., where fertilizer was applied) rate fraction when fertilizer had been added and an accelerated rate of utilization of the C20 to C25 group of n-alkanes when bacteria had been applied to the plots. DISCUSSION The hypothesis that a nutrient-deficient con- suggests that psychrophilic conditions prevail in the soils in this area. It has been noted (2) that these compounds are more recalcitrant to microbial attack under psychrophilic than under mesophilic conditions. The slight difference in the chemical composition of the oil recovered from soil and that obtained from the barrel was probably a result of our extraction procedure. However, the procedure, at least for the major components of oil (i.e., saturates and aromatics) gave very highly reproducible results (Fig. 1). Initial studies were performed comparing n-pentane, benzene, methanol, and methylene chloride as extractives for recovering oil from soil. All of these solvents readily recovered the n-saturate fraction of oil applied to soil, but only n-pentane did this without removing residual soil material which subsequently interfered with our analytical procedures. For example, the material extracted from the soil by the other solvents was subsequently extracted into the benzene fraction and thus effected the further purification of the oil. The increase in the insoluble fractions when fertilizer had been applied to the plots (for example, the insoluble asphaltenes) suggests that transformation of oil components is taking place along with the assimilation of the n-satu-rate fraction. Changes in polarity of components result from the introduction of oxygen into the various components. Since our liquid chromatographic separation procedure is based on polarity, this change would shift these compounds into more polar fractions. At this time, however, it is not possible to account for the increase in total asphaltenes which were detected in the oil recovered from the plots 308 days after treatment. The lack of stimulation of the mold population as determined by our plate count technique is possibly an artifact resulting from our procedure, or more likely it reflects the inability of molds to successfully compete with bacteria under the nonrestrictive environmental conditions found in our plots. The slight increase in the rate of oil utilization by the application of bacteria to the spills could be a result of too low a level of application or to the inability of these bacteria to survive under the natural field conditions existing in the Swan Hills area. However, considering that nc'C FIG. 4. Gas chromatographic profiles of the saturate fraction of the Swan Hills oil 308 days after contact with the soil and various amendments. the bacterial content of these soils is 10-fold higher than what we applied, the failure to obtain a clear-cut effect is probably a result of too low a level of application. The partial utilization of n-saturates observed 308 days after application in those plots where oil alone was applied indicates that some component(s) of the indigenous flora present in the Swan Hills soils has the capability of utilizing crude oil. Visual observation of the rate of recovery of plant growth indicates an accelerated rate of recovery for those plots which received fertilizer. Thus, an increase in bacterial numbers can be correlated with an increased rate of disappearance of the n-saturate fraction component of crude oil, and this latter observation can be correlated with an increased rate of recovery of vegetation. All of these effects can be directly related to the application of fertilizer (ureaphosphate) to oil-soaked plots.

v3-fos

2018-04-03T00:26:23.239Z

1974-10-01T00:00:00.000Z

11957375

s2

Concentration of Enteric Viruses from Water with Lettuce Extract A method for recovering enteroviruses, adenovirus, and reovirus from water with lettuce extract is described. Lettuce extract at pH 8.5 was added to the sample and the pH was reduced stepwise with hydrochloric acid to 4.0 to 4.5. The flocculent lettuce-extract particles, and adsorbed virus, were readily removed from solution by low-speed centrifugation. Electron microscopy suggests that, under conditions suitable for adsorption, virus particles are coated with the lettuce-extract colloid. A number of methods for the recovery of extended to include other sample and the pH was reduced stepwise with hydrochloric acid to 4.0 to 4.5. The flocculent lettuce-extract particles, and adsorbed virus, were readily removed from solution by low-speed centrifugation. Electron microscopy suggests that, under conditions suitable for adsorption, virus particles are coated with the lettuce-extract colloid. A number of methods for the recovery of virus from water have been described recently (1,(4)(5)(6)(7). Virus adsorption followed by elution has been the principal approach for virus concentration. In an earlier report (2), we described a method for enterovirus recovery using lettuce floc. The work has been extended to include two other enteric viruses, reovirus 1 and adenovirus 7a. Lettuce extract was prepared as described previously (2). The extract is a clear amber-colored colloidal suspension at pH 5.5 or above. A floc forms below pH 5.5 and is readily sedimented by low-speed centrifugation at pH 4.0 to 4.5. The dry weight of the floc varied from 1.5 to 3.0 mg/ml depending on the batch. Approximately 50% of the dry weight was protein, as determined by the method of Lowry et al. (3). Adsorption to lettuce extract of coxsackievirus types B4 and B5, echovirus type 7, poliovirus type 1 (Sabin), reovirus type 1, and adenovirus type 7a was examined as follows. Virus concentrations of 100 or 1,000 plaqueforming units (PFU) in 0.1 ml of phosphate-buffered saline were added individually to samples containing 10 to 1,000 ml of distilled water. Final virus concentrations varied from 0.1 to 100 PFU per ml of sample. A similar inoculum was added to duplicate samples of 2.5 ml of growth medium to serve as virus controls. A 10% volume of lettuce extract was added to the water samples at pH 5.0, 6.0, 7.0, 8.0, or 8.5. Samples were adjusted to pH 4.0 to 4.5 in 0.5 to 1 log steps by dropwise addition of HC1. After centrifugation at 1,000 x g for 10 min, the pellets were dissolved by adding NaOH; 0.05 ml of 1 N NaOH dissolved the 0.4to 0.8-ml pellet obtained from a 200-ml sample. Water samples larger than 200 ml were divided, centrifuged, and finally recombined after dissolution of the pellets. The sample was diluted to 2.5 ml with concentrated medium 199 to give single strength medium and then was assayed on a single monolayer of cultured cells as described previously (2). HEp-2 cells were used for the coxsackievirus and poliovirus, Vero cells were used for echovirus, and primary African green monkey kidney cells were used for reovirus and adenovirus. Enterovirus plaques were read after 3 days, adenovirus and reovirus after 8 days. Results of preliminary experiments indicated that virus in the dissolved pellet alone or with added 10% serum would not adsorb to cell monolayers. Incorporation of medium 199, Earle, or saline with or without 10% serum, however, permitted infection of monolayers. Coxsackievirus B4, B5, echovirus 7, poliovirus 1, and adenovirus 7a were efficiently concentrated from water with colloidal lettuce extract at pH 6.0 or higher. Flocculent extract at pH 5.0 was less efficient in concentrating adenovirus than enteroviruses; reovirus required a pH of 8.0 for effective concentration (Fig. 1). Quantitative recovery of all viruses tested was accomplished by adding lettuce extract at pH 8.5 to the water sample followed by the dropwise addition of HCl in 0.5 to 1 log steps to pH 4.0 to 4.5. Virus inputs varying from 0.1 to 100 PFU per ml of sample were concentrated from water volumes of 10 to 1,000 ml. Concentrates from volumes as great as 500 ml could be assayed in one plastic dish without apparent toxicity to the cell monolayer. Less than 1% of the virus input remained in the supernatant fluid as unadsorbed virus. Mixed as well as unmixed populations of reovirus and adenovirus were adsorbed. For electron microscopy, viral suspensions were diluted with 2% potassium phosphotungstate at pH 6.8 and spread on pure carbon or carbon-coated Formvar electron microscope grids. The carbon surface was rendered hydrophilic by a brief treatment (ca. 40 s) of exposure to ionized air in a Plasmod unit (Tegal Corp., Richmond, Calif.). The negatively stained viruses were then examined at a magnification of 60,000 to 100,000 in a Siemens Elmiscop 101. Electron photomicrographs showed that virus was coated with colloidal particles or aggregates of them at optimal pH levels. Capsomers of reovirus were sharply defined at pH 6.0, but were indistinct at pH 8.5 due to adsorbed lettuce-extract colloid (Fig. 2). Adenovirus was well defined at pH 4.5 but hazy at pH 6.0 (Fig. 3). Controls of reovirus and adenovirus without lettuce extract at similar pH measurements were all sharply defined. Colloidal particles were a few nanometers in diameter; some aggregates of the particles were as large as 100 nm. By following the procedure outlined above, enteroviruses, adenoviruses, and reoviruses are efficiently removed and concentrated from water.

v3-fos

2020-12-10T09:04:12.306Z

1974-05-01T00:00:00.000Z

237231925

s2

Microculture System for Detection of Newcastle Disease Virus Antibodies A microculture system utilizing cytopathic effect (CPE) and hemadsorption (HAd) end points was effective in determining the level of Newcastle disease virus (NDV) antibodies. The microculture system was of comparable sensitivity to the plaque reduction test for the detection of NDV antibodies. The standards by which the CPE and HAd microculture tests would be considered reproducible were defined. The results indicate that the CPE and HAd microculture tests are reproducible within one twofold dilution. In recent years, microculture methods for virus titration and serological procedures have come into more frequent use. The application of microculture has been reported in the study of arboviruses (2), transmissible gastroenteritis virus (11), rubeola virus (6), poliovirus (5), respiratory virus seroepidemiology (8), and in other serological investigations (3,4,9). Methods utilizing microculture have been shown to be as sensitive and far more economical than macro cell culture methods (4). Laboratory procedures used to detect Newcastle disease virus (NDV) antibodies include the hemagglutination-inhibition (HI) test, neutralization in embryonated eggs, plaque reduction, and the egg-bit technique (1). The detection of HI antibodies and virus neutralizing (VN) antibodies has been utilized as an indication of exposure to NDV. Serum neutralization tests are universally accepted as standard quantitative tests for antibody levels. However, the neutralization tests for NDV, by using either embryonated eggs or cell cultures as a virus indication system, are expensive and cumbersome. The objective of this study was to develop a virus neutralization test for NDV antibodies utilizing microcultures as the indicator system for unneutralized virus. MATERIALS AND METHODS Media. Growth medium for cell cultures consisted of Hanks balanced salt solution supplemented with 0.25% lactalbumin hydrolysate, 10% fetal calf serum, and 10% tryptose phosphate broth. Penicillin and streptomycin were added at concentrations of 100 U/ml and 100 Ag/ml, respectively. Virus dilutions were prepared in Hanks balanced salt solution. Agar overlay medium, for plaque enumeration, consisted of Eagle minimal essential medium supplemented with 2% fetal calf serum and 1% purified agar (Difco Laboratories). Cell cultures. Chicken kidney cell cultures were used for virus propagation. Kidneys from 1-day-old chicks were trypsinized for 30 min, and the dispersed cells were filtered through sterile gauze and sedimented by centrifrigation. A 1-ml amount of packed cells was suspended in 200 ml of growth medium. Source of virus. The Kansas-Manhattan strain of NDV was supplied by P. D. Lukert, University of Georgia. This strain was selected because of its rapid cytopathic effect (CPE) in chicken kidney cell cultures. Serums. Serums used in the experiments were obtained from 34 individual chickens. Thirty serum samples were obtained from broiler chickens in northern Georgia. The broilers were given NDV, B-1 vaccine at 7 days of age by aerosol; serum samples were collected 42 days later. Four negative serum samples were obtained from unvaccinated specific pathogenfree chickens housed at the Poultry Disease Research Center, University of Georgia. To avoid bias, all serum samples were randomly coded before each test. In this manner, the sera were examined in a sequence unknown to the individual conducting the tests. The titration end points were not decoded until the data were ordered for statistical analysis. Virus-neutralization tests: plaque reduction. Neutralizing activity of sera by plaque reduction was determined by the constant virus serum dilution technique. A virus dilution shown to give approximately 100 plaques was mixed with an equal volume of each serum dilution. Plaques were counted after incubation for 48 h, and the highest serum dilution that gave greater than 50% plaque reduction as compared with virus control plates was recorded as the end point. 390) Co., New Haven, Conn.) were used for determination of virus neutralization by inhibition of viral CPE. Twofold serum dilutions from 1: 10 to 1: 1,280 were prepared in tubes. The serum dilutions were added to equal volumes of NDV so that each 0.05 ml of virus-serum mixture contained 1,000 to 2,000 plaqueforming units (PFU) of NDV. The virus-serum mixtures were mixed in a test tube and allowed to react for 30 min at 26 C. Microculture plates were inoculated with 0.05 ml of the virus-serum mixtures. Each virus-serum dilution was inoculated into eight replicate well cultures. Five uninoculated replicate well cultures served as cell controls. Virus controls consisted of inoculating five replicate well cultures (Fig. 1). After the inoculation of the virus and virus-serum mixtures, each well was inoculated with 0.15 ml of the chicken kidney cell suspension. The microculture plates were then sealed with a clear mylar sheet with an adhesive back (35 PSM, Linbro Chemical Co., New Haven, Conn.) and covered with a clear polystyrene top (53, Linbro Chemical Co., New Haven, Conn.). The microculture plates were incubated at 37 C for 48 h. The incubation period, temperature of incubation, and PFU of NDV utilized in the test were determined by preliminary trials to obtain maximal sensitivity in the shortest period of time. After incubation, the medium was removed, and the cells were fixed with 10% neutral Formalin for 3 to 5 min. The Formalin was removed, and the fixed cells were stained with 1% crystal violet for 30 min. The stained cells were examined by gross inspection with an appropriate light background. Control monolayers and virus negative monolayers appeared solid blue. Virus-infected monolayers were mottled or had discrete plaques (Fig. 1). The antibody titers obtained in the microculture CPE system were calculated by the Kiirber method (7). Microculture hemadsorption. Hemadsorption (HAd) tests were conducted in the same type of plates used for the microculture CPE test (Fig. 1). In this case, 0.05 ml of chicken erythrocytes were added to three replicate cultures which had been inoculated with virus-serum mixture, to five replicate cultures used for virus controls, and to five replicate cultures used for cell controls. The red blood cells were allowed to settle at 4 C. The wells were washed two times with phosphate-buffered saline and examined microscopically for HAd. The antibody end points were determined as the reciprocal of that serum dilution which completely inhibited the adsorption of chicken red blood cells. Reproducibility of antibody titers in microculture systems. The reproducibility of antibody titers obtained in the microculture systems was determined by repeating the microculture CPE and HAd tests with 25 of the serum samples three times at intervals of 4 months and 1 week. Statistical analysis. Assessment of the accuracy of the CPE and HAd microculture tests requires a knowledge of the true neutralizing antibody titers in CPE and HAd microculture systems. This information is not available. Therefore, the question of accuracy cannot be answered directly. However, if results of a test system are consistently reproducible, accuracy can be inferred. Because the true CPE and HAd microculture titers remain the same, only the variations (human and mechanical) inherent in any test system need to be considered to answer questions concerning reproducibility. The mean and the standard deviation are the "best" estimates of variation (10). By utilizing these statistics a "reproducibility" level can be defined. An acceptable level of reproducibility of many serological tests is, by custom, commonly referred to as being "within" one twofold dilution. By employing the statistics which can be developed and the presently accepted custom, two hypotheses are advanced. (i) The CPE and HAd microculture tests are reproducible between one twofold dilution (Fig. 2). (ii) The CPE and HAd microculture tests are reproducible within one twofold dilution (Fig. 2). The criterion of reproducibility revolves around "between" and "within" one twofold dilution. The logarithmic range between one twofold dilution is 0.602 and within one twofold dilution is 1.204 (Fig. 2) 27,1974 confidence interval of the means of the CPE and HAd microculture titers do not exceed 0.602 (between) or 1.204 (within). From the logarithmic numbers obtained from the end point determination, the mean, standard error, coefficient of variation, and confidence interval for the CPE and HAd microculture titers of each serum sample were calculated (10). The confidence interval based upon the t-distribution was calculated at the 90 and 95% levels. RESULTS The neutralizing antibody end points in microculture CPE and HAd systems were comparable to those obtained by the plaque reduction technique. The results are shown in Table 1. The reproducibility of the microculture CPE and HAd tests was established by utilizing the t-distribution. Confidence limits for serum sam-APPL. MICROBIOL. ple antibody titer means were calculated at the 90 and 95% levels. At the 90% confidence interval, it was ascertained that the range for the CPE microculture test did not exceed the standard of reproducibility of being between one twofold dilution in 17 of the 25 sera tested. Additionally, all of these confidence intervals were within one twofold dilution (Table 2). It was also determined that at the 95% confidence interval this range for the CPE microculture test exceeded the standard of reproducibility of being between one twofold dilution in 10 of the 25 sera. However, when the standard of reproducibility was to be within one twofold dilution, only 3 of the 25 sera failed to meet this standard. The coefficient of variability is another method of describing variation in a population. In this case, in the three CPE microculture titers of each serum sample, the average coefficient of variability was 6%. It should be noted that sera 6, 24, and 27 had coefficients of variability of 12.7, 9.9, and 16.1%, respectively. These were the three largest coefficients of variability, and in each case the confidence interval (95%) exceeded the standard set forth for reproducibility ( Table 3). Nine of the sera examined in the HAd system had the same titer in each of the three trials. Each of the remaining 16 sera examined had two identical titers, and the third differed by only one twofold dilution (Table 1). Of the sera which had the same titers for each trial, the standard error of the mean titration was, of course, zero and all met the reproducibility levels at any of the standards previously described. For the other 16 sera the calculated logarithmic standard error of the mean was 0.1003. Therefore, the confidence interval at the 90% level was 0.2935 and the confidence range was 0.5870 (see legend, Table 2). At the 90% confidence level these sera did not exceed the reproducibility standard either between or within one twofold dilution. Similarly, the confidence interval at the 95% level was 0.4316, and the confidence range was 0.863 (see legends, Tables 2 and 3). At the 95% level, all of these sera failed to meet the reproducibility level of being between one twofold dilution, but all met the standard of being within one twofold dilution. The average coefficient of variation of the 25 titers in the HAd system was 4.8%. DISCUSSION The results presented indicate that the microculture CPE and HAd test systems were of comparable sensitivity to the 50% plaque reduction test for detecting neutralizing antibodies to NDV. Plaque reduction end points were read as that serum dilution that decreased the plaque numbers greater than 50% as compared with the virus control count. The microculture CPE antibody end points were determined by the KArber method, in which titers are interpolated between the serum dilutions showing greater than and less than 50% of the virus control CPE. Antibody end points in the HAd system were determined as that serum dilution which completely inhibited the adsorption of chicken red blood cells. The small variation in the sensitivities of the microculture CPE and HAd tests and the plaque reduction test may be due to the method of reading the end points. The specificity of the microculture system was tested by the blind inclusion of four sera derived from specific pathogen-free chickens, in which cases all tests were negative. The microculture method, in addition to being very sensitive in detecting NDV antibodies, has the advantage of being economical, because the quantity of kidney cells harvested from a single 1-day-old chicken is sufficient to establish microcultures in approximately 300 microculture wells. Because CPE are not measured by microscopy examination, but by gross visual inspection of plates, the microtiter CPE method requires less time, and there is less chance for subjective error in reading CPE end points. It was stated earlier that knowledge of the true CPE and HAd microculture titers of any serum is not available. The probability statements state that we are either 90 or 95% confident that we have included the true mean within the confidence limits outlined. For the CPE microculture test to be considered reproducible, the standard is that the calculated confidence interval range must either be between or within one twofold dilution. At the 95% confidence interval range, 22 of the 25 confidence interval ranges fell within one twofold dilution. This assessment of reproducibility seems rigorous since only three replicates were employed in arriving at a sample mean and standard deviation. Significant variation in any one of the calculated CPE microculture titers was enough to extend the confidence interval beyond the criteria stated to be acceptable as being reproducible. It would appear then that the human and mechanical variation inherent in any serological test is minimal in the CPE microculture test. This is further borne out by the calculated coefficients of variation which describe the amount of variation in a sampled population. The average titer variation of the sera sampled utilizing the CPE microculture test was 6%. Because individual investigators have varying requirements for reproducibility levels, no criteria for accepting or rejecting the two hypotheses stated earlier were delineated. However, in the case of the CPE microculture system, it would seem that the first hypothesis concerning reproducibility between one twofold dilution should be rejected. In this case, the calculated confidence interval range at 90 and 95% exceeded the reproducibility standard in 8 and 15 sera, respectively. The second hypothesis concerns reproducibility within one twofold dilution. At the 90% level, the confidence interval met the reproducibility standard in all cases. At the 95% level, the confidence interval met the reproducibility standard 22 out of 25 times. The chi-square criterion (corrected for continuity) was applied to determine if this result could have happened by chance alone. The calculated P of >0.005 made this seem unlikely. It would appear then that the second hypothesis can be accepted conditionally upon the standards of reproducibility required. For the HAd test, assessment of reproducibility was again rigorous because of the minimal number of replications employed. This situation is further complicated because the accepted method of measuring titers is crudely quantitative. Therefore, in a set of three titration end points, if two of the end points are the same and the third is different only by one twofold dilution, only the 95% confidence limits of being reproducible within one twofold dilution are easily met. Slight deviation of end points either sequentially (1: 20, 1:40, 1:80) or differing by a fourfold dilution (1: 20, 1: 20, 1: 80) lowers this estimation of the 90% confidence limits of being reproducible within one twofold dilution. These deviations did not occur, and it would appear that extraneous variation is minimal in the HAd test. This statement is supported by the average coefficient of variation of the titers which is 4.8%. At the 90% confidence level, the HAd test met the reproducibility standard in all cases. Therefore, the first and second hypotheses can be accepted if this level of confidence is acceptable. At the 95% confidence level, only 9 of the 25 sera met the reproducibility standard of being between one twofold dilution, whereas all were reproducible within one twofold dilution. At this level of confidence it would seem that the first hypothesis should be rejected and the second accepted. LITERATURE CITED 1. Beard, C. W. 1969. The egg-bit technique for measuring Newcastle disease virus and its neutralizing antibodies.

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2014-10-01T00:00:00.000Z

1974-05-01T00:00:00.000Z

14435313

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Significance of high soil lead concentrations for childhood lead burdens. The lead exposure of children and their mothers has been studied in two towns with mean soil lead contents of 900 and 400 ppm. No significant difference in blood or fecal lead contents was demonstrated between the two populations, but a small difference in hair lead content was shown. The blood lead content of children was greater than that of their mothers and was higher in the summer than in the spring samples. Children with pica for soil in the control area had increased lead content of blood and hair. Preliminary data for children and mothers from villages with mean soil lead contents of 500 ppm and 10,000 ppm are reported which show significant differences in blood and hair lead content within the normal range. The data suggest that soil lead content of 10,000 ppm may result in increased absorption of lead in children, but to a degree which is unlikely to be of biological significance. There has been increasing interest in the potential hazard to children resulting from the ingestion of soils or dusts containing concentrations of lead above normal, originating both from natural geological sources and from contamination resulting from industrial, mining, and smelting activity, as well as from lead additives in gasoline. The importance of this source of lead to childhood lead burdens is unknown, although at least one case of lead poisoning due to soil ingestion has previously been reported (1 A lead concentration of 15 ppm in soils is considered normal for virgin surface soils (2), but it can vary greatly, depending on the type of soil. Normal lead concentrations of less than 20 ppm have been reported in soils derived from sandstone, of 80 ppm in soils from quartz mica schist (3), and of 200 ppm in soils derived from black shale (4). Concentrations of several thousand ppm have been reported in city soils and dusts (1,5,6) which may, however, be "normal" for certain highly mineralized areas, such as that of the carboniferous limestone area of Derbyshire, which has been mined for lead and zinc since the Roman occupation of England. Lead poisoning in cattle grazing in Derby- May 1974 75 shire has been reported (7); this may have resulted from the ingestion of soil rather than high lead content pasture. Cattle have been shown to ingest large arrmounts of soil while grazing and may thus ingest up to ten times the amount of lead in the form of soil to that in herbage (8). A regional geochemical survey of the area, undertaken by the group at Imperial College, revealed many stream sediments with lead concentrations exceeding 3000 ppm lead and surface soils near old mines and smelter sites containing up to 3%o lead (9). On the basis of this survey, a study was undertaken in two towns differing in the lead content of their soil and located in a specific geochemically defined area in order to determine the significance that increased concentrations of lead in soil may have for local children. A similar study has recently been initiated in certain villages in the area which were found to have a mean soil lead content of approximately 10,000 ppm. Sampling Population samples in the two towns, Matlock and Buxton, were obtained through the local health authority and studied during April and May 1972. Children aged 2-3 years were chosen, since the prevalence of pica is high in this age group (10). Interviews were conducted at a local clinic where the mother was questioned about the pica history of the child, where the child played, and whether the family grew or used locally grown produce. They were specifically asked if their child chewed or swallowed paint or soil, or mouthed soil-contaminated toys and fingers while at play. Capillary blood samples were obtained from both mother and child in order to provide an index of short-term lead absorption. Dietary intake and intermediate exposure were estimated by a single fecal collection (11) and a sample of hair from the child, respectively (12). The same mothers and children were seen again during July and repeat samples obtained. After visiting the individual homes, 30 children in Matlock and Buxton were found to have little or no soil in their gardens or to live in streets with higher than average traffic density for the area. Since these children did not normally come in contact with soil or might have had greater exposure to airborne lead than the majority of children in our study, they were excluded from further data analysis. The lead content of the dust and rainfall and the suspended particulate matter in each town was monitored and soil samples taken from the home of each child. Six to ten subsamples, at a depth of 0-5 cm were combined to give surface soil samples from the front and back garden flower beds, the back garden lawn and, if present, the vegetable garden. A single sample was taken from the lawn at a depth of 30-45 cm. All samples were taken at least 2 m from the house. In 1973 a similar study was initiated in neighboring lead-contaminated and control villages. Venous blood samples were taken from children aged 2-5 years and their mothers, as well as the hair and stool samples from the children. Samples of garden soil and house dust were obtained from each home. Analysis Capillary blood samples were analyzed by atomic absorption by using a Perkin-Elmer graphite furnace HGA-70, with a Model 305 spectrophotometer. Venous blood was analyzed by atomic absorption by use of a Delves cup procedure. At least three replicate analyses were performed on each sample. The hair was washed with a nonionic detergent (Triton X-100) and the 2.5-cm segment adjacent to the scalp taken to indicate exposure over the previous two months (13). Stool samples were homogenized with distilled water and air samples collected on military filters (0.8 um), equipment conforming to British Standard 1747 being used. The hair, feces, dustfall and air filters were wet-ashed with nitric and perchloric acids and analyzed by a semiautomated dithizone procedure. Environmental Health Perspecfives Soil samples were oven-dried at 100°C, sieved to remove material greater than 2 mm, ground to less than 200 jam, and digested with nitric acid before analysis by flame atomic absorption spectroscopy. Results Soil lead data are summarized in Table 1. In addition to soils from the individual homes, data are presented on soils from grasslands in and around each town. The geometric mean is given, as the soil and biological data followed a lognormal distribution. The values in the control town, Buxton, were greater than expected from the stream sediment survey, but there was at least a factor of two between the mean soil lead contents of the two towns. For each type of soil sample, the mean value in Matlock was significantly greater than the mean in Buxton (two tailed t-test, P <0.05). The subsoil and topsoil lead concentrations of Matlock grassland sites were similar, indicating that the high soil lead concentrations in that area were probably due to mineralization. The monthly dust and rainfall and the suspended particulate matter lead concentrations were found to be within the range for similar areas in England (14). For the year beginning June 1972, the mean lead concentration in suspended particulate matter was 0.61 /Lg/m8 in Matlock (range 0.33-1.06) and 0.29 /Lg/m3 in Buxton (range 0.12-0.47). Although there was a difference in concentration, the low values indicate that airborne lead was not contributing significantly to lead exposure in either of the two towns. The blood lead data are summarized in Table 2. There was no significant difference in the mean blood lead concentrations of the children or mothers between the two towns, although the mean surface soil concentrations of 909 ppm in Matlock and 398 ppm in Buxton were significantly different (P <0.05). Both children and mothers were found to have greater blood lead concentrations in the summer than the spring, and the children had higher blood lead concentrations than their mothers (paired t-tests, P <0.05). This applied to children with or without current pica ( Figs. 1 and 2). In each town, comparisons were made between the children who had no current pica and those who had current pica for anything, and those who had current pica for soil (Table 3). In Matlock there was no significant difference in the mean blood or fecal Our definition of pica for soil included those children who habitually put fingers or toys in their mouths while playing in their gardens, as well as actually putting soil directly into their mouths, and included known by their mothers to have swallowed soil. lead values for these groups in either the spring or summer sampling. In Buxton, the only significant difference (P <0.05) was found for the summer sample, where the children who have pica for soil had a greater mean blood lead concentration than the children who had no current pica. Although soil-eating children had a greater mean blood lead compared with those who had no current pica, the corresponding mothers also showed a similar difference ( Table 4), suggesting that the increased blood lead was due to factors other than soil. This finding illustrates the value of also Environmental Health Perspectives sampling the mother as a further indication of the environment of the child. The hair lead results provided no conclusive evidence of an effect of soil lead on childhood lead exposure (Table 5). While in Buxton the children with pica for soil had a statistically greater mean hair lead concentration than those without a current pica history, the values are low and within the normal range. The children in Buxton with current pica for anything also had a greater mean hair lead concentration but no comparable difference was demonstrated in Matlock, the town with the greater soil lead content. The individual results were related to the soil lead values found at each child's home by product-moment correlations of the raw and log-transformed data, as well as the nonparametric Kendal rank correlation. No statistically significant correlations between the blood, fecal, or hair lead values and the soil lead levels in the immediate environment of the child were found by these methods. Further studies are currently being conducted in two groups of villages near Matlock and Buxton, where the mean soil lead concentrations are approximately 10,000 ppm and 500 ppm. The homes with the higher May 1974 79 lead soil concentrations are in villages near extensive old mine workings and some are, in fact, built on old waste material. The blood and hair analyses have been completed, and the preliminary data are given (Table 6 and Fig. 3 and 4). The children were divided into the hiigh and low soil lead areas on the basis of soil samples taken prior to the survey, and any final interpretation must await the analysis of the soils from the individual homes. The range of the values found were 13-45 pug/1OO ml for the children's blood, pfg/100 ml for the mothers' blood, and 2-62 ,ug/g for the hair samples. All children and mothers living in the high soil lead area had greater mean blood and hair levels than those living in the low soil lead area, as well as those children with no current pica history. Although these differences are significant at the 95%o confidence level, it should be noted that all the observed values were within the accepted normal range. Comparison of the data for children with present pica for soil and those with no current pica showed that only the hair results in the high soil lead area were significantly different. The 16 children with pica for soil had a mean hair lead concentration of 21 ppm compared with a mean of 11 ppm for the 27 children without current pica (P <0.05). There was no statistically significant difference in the mean blood lead concentrations. Further data analysis will take into account any age differences and the results of the analysis of the garden soils and house dusts from the individual homes. Discussion While the data suggest that there is increased lead exposure for children living in high soil lead areas, there is no evidence that this is sufficient to be of biological significance. Pica for soil, although prevalent to an unexpected degree, appears to be a relatively unimportant source of lead for children. This could be due either to the small amounts of soil being ingested or the relatively poor bioavailability of lead in the ingested material, similar to the poor uptake of lead by plants (15). Since soil type may greatly influence the bioavailability of lead in ingested soil, the control villages for the study in progress were carefully selected to represent similar soil parent material with a relatively low lead content. Further studies on the significance of lead in soil and dusts for childhood lead burdens are in progress. The amounts of soil ingested by children and the factors which may influence the availability of lead from various types of soil and city dust are being investigated by tracer, leaching and animal feeding studies. The results of our studies to date suggest that local soil lead levels of the order of 10,000 ppm are without major significance and that on present evidence the recent concern with regard to contaminated soils in cities is not well founded. Security, and the Natural Environment Research Council. D. B. is a Wellcome Senior Fellow in Clinical Science.

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2009-02-12T00:00:00.000Z

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Causes of variation in the frequency of monozygous and dizygous twinning in various breeds of cattle 1974. Causes of variation in the frequency of monozygous and dizygous twinning in various breeds of cattle. A statistical investigation has been made comprising 12 European cattle breeds with a total of about 5.3 milj. calvings and 120,000 twin births. Season of conception has a marked influence on the frequency of twin births, one maximum corresponding to spring and another one to autumn conceptions. There are significant breed differences in the relation between age of the cows, measured in parities, and the frequency of monozygous (MZ) as well as dizygous (DZ) twin births. On an average, the DZ frequency increases more than the MZ frequency from the first to the fourth-fifth calving; then the age-curves show a plateauing tendency. After the first twin birth, the twinning frequency at the following calvings is 4-5 times higher than in the general population. Apparently, twinning depends on polygenic inheritance and also on various environmental influences, with a threshold for the phenotypic manifestation. It may be assumed that the underlying liability of the cows in a population to bear twins has an approximately normal distribution. The heritability of twinning has been estimated on the binomial p-scale as well as on the x-scale of the normal curve. On the p-scale the heritability of twinning was found to be about 0.02 which corresponds to 0.16 on the x-scale, although with some differences between breeds. In 1932, the senior author published a treatise on "The sex ratio and multiple births in cattle", based mainly on data from a number of pedigree herds of Swedish dairy breeds. The herds were selected in order to obtain as accurate records as possible of all the calvings within the herds, without exclusions of any kind. Previous as well as more recent investigations by numerous authors have shown that herdbook data are, as a rule, unreliable for this kind of study, because of numerous omissions of cases of twinning, and the sex of calves, in the reports of breeders to the herdbook offices. With few exceptions, the investigations on twinning in cattle where this source of error has been recognized, have therefore been limited to some large herds where careful notations have been made on the reproductive performance of all the breeding animals, but such data are usually too limited for detailed studies of the problems involved. However, with the widespread use of artificial insemination, milk recording, and electronic dataprocessing, this situation has changed, at least for some types of investigations. Large amounts of data are now available for studying such effects as the influence of mating season and age of the cows on the twinning incidence, as well as the proportions between monozygous (MZ) and dizygous (DZ) twin births. The first estimate of the frequency of MZ twins was made on data from various sources (Jo-HANSSON 1932) with the result that 11.6 % of all like-sexed twin pairs were monozygotic. Neither the effect of breed nor age of the dam was considered. The age of the cow at the time of conception has a pronounced effect on the likelihood of her producing twins. Breed comparisons can give reliable results only when the age distribution of the cows on parities is taken into consideration. This is particularly important as regards the total frequency of twinning but it also seems to be important for the estimation of the ratio of MZ to DZ twinning incidence. A new investigation of the twinning problems, based on large numbers of reliable and unselected data, would therefore appear to be justified. The present study of twinning in cattle does not include the relation of twinning to milk yield or to the reproductive functions of the dams. These problems have been discussed recently in a comprehensive review by HENDY and BOWMAN (1970). The major topic considered here is the causes of variation in the frequency of MZ and DZ twinning. Material and methods The data on twinning in the Swedish breeds were obtained from the Swedish Association for Improvement of Animal Production (SHS), and the main tabulations of these data have been made at the Agricultural Center for Data Processing at HAllsta. The data for the Danish breeds emanate from the publication "Kaelvningsstatistik" by ELLEBY and MYGIND-RASMUSSEN (1971), although additional calculations from their tables have been made by the present authors. ELLEBY (1973) has provided some "fresh" data on twinning and triplet-births. Data for dairy breeds in South Germany (Bavaria) and in Niedersachsen have been placed at our disposal by the courtesy of Dr. H. Schumann, director of the "Landeskuratorium der Erzeugerringe fur Tierveredelung in Bayern" and by "Rechenzentrum fur Forderung der Landwirtschaft in Niedersachsen". We have also obtained data on the Swiss Simmental cattle from Mr. Claude Gaillard, director of the "Schweizerischer Verband fur kunstliche Besamung". Furthermore, data were obtained from a large breeding establishment for Hereford cattle in the U.S. A., viz. the Wyoming Hereford Ranch, Cheyenne Wy. With regard to the Charolais breed in France we have obtained permission to quote some data from an unpublished manuscript by Mr. Menissier, DCpartement de GCnCtique Animal, Jouy-en-Josas. A small amount of data were also available in Sweden on the British beef breeds Hereford and Aberdeen-Angus and on the French Charolais, which are now used in Sweden and other Northern countries for crossing with dairy breeds for beef production. Practically all data for the European breeds relate to the period 1968-72. The methods used in analysing the data are simple and follow traditional lines, employed in human genetics. However, the methods used in estimating the fraction of the total number of twin pairs which are probably monozygotic, and the standard deviation of the estimates, may need some discussion. As early as 1902, the German physiciangeneticist WILHELM WEINBERG devised his "differential method" for estimating the number of MZ twin pairs within a random sample of twins where the sex ratio was about 50 % males. The probability for the male and female sex would then be equal (p=q=O.5), and with random distribution of the sexes within pairs, the distribution of the three possible sex combinations would be (p +q)2 =0.2563 +0.506? +0.259?, i.e. equal numbers of like-sexed and unlike-sexed pairs. An excess of like-sexed pairs may then be used as an estimate of the number of MZ pairs in the sample. Where the number of twin pairs = n, the number of like-sexed pairs = n, and the number of unlike-sexed pairs = n,, the estimated number of MZ pairs would be n -2n2. Because of the importance of MZ cattle twins for various types of experiments, their frequency is often expressed as a fraction (or percentage) of the number of like-sexed twin pairs: n -2n, n1 m = ~~ BONNIER (1946) preferred to use the observed sex ratios in the twin sample rather than the approximate ratio of 50 % males, thus estimating the value of m = ~~~ 2 p q n -n 2 . However, the gain 2 w h -n d . . in accuracy by introducing the term 2pq, where p and q are the observed frequencies of males and females in the twin sample, is very slight, as pointed out by MEADOWS and LUSH (1957). The intra-uterine and perinatal mortality, which is known to be selective against males, is much higher for twins than for single born animals, and the sex ratio of twins is seldom higher than 52 % males (cf. Table 3). With this ratio, the term 2pq=0.4992 instead of 0.50 for equal numbers of males and females. It is important, however, to define the stage at which the sex of the twins is determined. The secondary sex ratios of single born calves and twins, presented in Table 3, refer to calves born alive at full term. However, there may be some differences between countries in applying this general rule. It may be added that not only is the fraction m of interest in a twin study, but also the relation between the number of MZ pairs to the DZ's and to the total number of births. In calculating the standard deviation of twinning percentages (pt) we use the formula spt = ljpt(lmpt), where n = the number of calvn __ ings on which the twinning percentage is based. However, when calculating the standard deviation of an estimate of the frequency of MZ twins, it must be borne in mind that this estimate is based on the fractions of like-sexed and unlikesexed twin pairs in the population, and each of these two samples has its sampling error. BONNIER (1946) derived the following formula for the standard deviation of the calculated fraction (m) of MZ twins out of like-sexed twins: sm= 2(1-2pqm) v T . Since 2pq can be eliminated without any noticeable loss of accuracy, the formula can be simplified to sm = (2 -m) -The method used in estimating the heritability of the twinning tendency will be discussed later (p. 219). . Perinatal mortality and the sex ratio of twins Perinatal mortality and the secondary sex ratio of twins, compared to single born calves, are of interest from a breeding point of view, and they are important in estimating the relative frequency of MZ and DZ twinning. Our data relating to these problems are meager, but they will nevertheless be presented in order to illustrate the situation within some of the breeds studied. It is well known from human genetics that the sex ratio of aborted and stillborn fetuses is and single born calves considerably higher than that of children born alive at full term (cf. STERN 1960). CHAPMAN et al. (1938) studied the sex ratio of cattle fetuses at different stages of development using slaughterhouse material. The data, comprising 2,044 fetuses on which the crown-rump length was measured, were grouped into ten classes with an interval of 10 cm. The material did not show a gradual decline in fetal sex ratio from the first to the tenth class but a rather sudden drop from the smallest fetuses with less than 10 cm crown-rump length, where the sex ratio reached 66%, to 56 % in the next class (11-20 cm) and then a further decrease to about equality in the 80-90 cm class. The early stage of high male mortality corresponded roughly to the third month of pregnancy. In the Swedish A1 and milk recording associations, abortion is defined as delivery of the fetus before 260 days of gestation. Stillbirth includes not only calves dead at birth but also those dying within a day after birth. Therefore, the term perinatal mortality will be used rather than stillbirth. Calf mortality within a month after the date of birth is called "early death". In practice, the sex of aborted calves is recorded only when abortion occurs during the later stages of pregnancy, and even here the records may be incomplete. The sex recording in perinatal mortality is fairly complete, although omissions may occur. The available data are examined and compared with similar data from Denmark, Germany and Switzerland. In Table 1, data are presented on the sex ratio for perinatal mortality of single fetuses in four different breeds. The sex ratio of aborted calves in the Swedish breeds SRB and SLB is close to 70% males, and the corresponding ratio for perinatal mortality is about 60 %. The sex ratio for "early deaths" is somewhat less than 50% which probably is due to an after-effect of the high sex ratio for perinatal deaths. The Swiss and German data in Table 1 are not quite comparable to the Swedish data, and therefore separate headings are used in the table. "Stillbirths" actually means here "dead at birth", and the figures for calves dying within four weeks include mortality during the first day after birth. This explains, at least partly, the high sex ratios for post-natal deaths. KRAUSSLICH (1972) has published data from 271,870 calvings of German Fleckvieh where he found the sex ratio for 3,769 Comparable data in connection with twin pregnancies are difficult to obtain. The results from a one year sample of abortions in the Swedish breeds (1971) are presented in Table 2, together with data on perinatal deaths from a larger sample of twin births. If the figures for perinatal mortality at twin births are compared with those for single births in Table 1, it will be observed that mortality was on average 4.3 times higher for twins than for single born calves in the Swedish breeds. For 721,180 single births in the Niedersachsen Schwarzbunt, the mortality rate was 2.8 % and for 14,773 twin births it was 13.1 %, or 4.7 times as high as for single births. The sex ratio at perinatal deaths of twins in the Swedish breeds was somewhat lower, however, than that for the single-born calves. This is probably due to a higher rate of abortions during twin pregnancies. Table 3 is designed to show the secondary sex ratio of single born calves and twins. The sex ratio of twins is expressed in two different ways, viz. for the total number of twin calves and for the like-sexed twin pairs only. Obviously, the sex ratio of unlike-sexed pairs is always 0.5, whereas the proportion of male and female pairs may vary to some extent. The figures in Table 3 do not indicate that the loss of male twin pairs is greater than that of female twin pairs at the time of delivery when the sex of the calves was determined. It may be concluded that Weinberg's method for estimating the incidence of MZ twins births is valid for cattle as well as for man. When comparing the sex ratios of different breeds, it is probably safest to make the comparisons only within the same organization for AI, milk recording, and data processing because the practice of sex recording may differ somewhat between the organizations. By doing so, it is interesting to find that the ratio is higher for the Red and White and the Danish Red (SRB and RDM) than for the Black and White (SLB and SDM) breeds. For single born calves the difference between SRB and SLB is 2.0k 0.038 % and between RDM and SDM 1.4f 0.028 %. With regard to twin births the sex-ratios are equal for the two Swedish breeds but there is a significant difference between the two Danish breeds (2.2 f 0.408 %) when calculated on the like-sexed pairs. One explanation for breed differences in the secondary sex ratio could be differences in intrauterine mortality and incidence of stillbirths. For the Swedish breeds there are some indications that these mortality figures are higher in the Friesians than in SRB (Table 1) but for the Danish breeds we know of no such indications. Seasonal variation in twinning frequency Only one environmental factor influencing the incidence of twinning can be studied from the available data, viz. the season of conception. No data are available on the nutritional level of the various herds, or of the individuals within the herds, but the milk records of the tested herds in Sweden and Denmark indicate that the plane of nutrition is on a n average fairly high. In Sweden, the average yield of 4 % fat corrected milk of all recorded cows in the year 1971-72 was for SRB 5,394 kg (221,465 cows) and for SLB 5,704 kg (76,978 cows). In earlier studies of twinning frequency in the Swedish breeds the senior author (JOHANSSON 1932) found two yearly peaks, one corresponding to conceptions in the early spring and another, more pronounced, after conceptions in September-October. The causes underlying these seasonal variations were discussed, and it was assumed that the temperature changes, causing changes in metabolism, were important factors. Later investigations on farm animals, especially on sheep, have demonstrated the effect of changes in the length of daylight in the autumn on the reproductive functions, and this would probably also apply to the bovines, at least to some extent. The present study of seasonal variations in twinning is based on a larger amount of data from five dairy breeds, viz. the Swedish Red and White cattle (SRB) and Swedish Friesians (SLB), the Danish Red (RDM) and Black and White (SDM) cattle, and the German Schwarzbunt in Niedersachsen, altogether nearly 3.5 million calvings and about 73,000 twin births (cf . Tables The diagrams in Fig. 1 show the seasonal distribution of calvings for each of the Danish and Swedish breeds mentioned, and for the German Schwarzbunt in Niedersachsen. The Swedish and Danish data could be classified into the first and the following calvings only, but the German data were completely classified on calving number. In all the populations studied, the calving incidence of primiparous animals reaches a maximum in the autumn, although somewhat later in Niedersachsen than in Sweden and Denmark. The seasonal distribution of calvings in Niedersachsen changes gradually with increasing age of the cows from an autumn to a spring maximum, but the greatest change occurs from the first to the second calving. All data for the fourth and following calvings have been pooled, since the change in distribution from one calving to the next is very slight. Table 8 in the publication by ELLEBY et al. (1971) shows that the change in seasonal distribution of the calvings with increasing age of the cows is very nearly the same in Denmark as in Niedersachsen, and it is also about the same in Sweden. Spring calving has been predominant in the Scandinavian countries for many centuries and it was probably the normal case among the aurochs. Feed was more scarce in winter than in summer, and the nutritional value of the feed was certainly superior in the summer. Under the present economic conditions, a relatively high incidence of autumn calvings is usually preferred. The mating of the heifers is timed by the breeders to result in autumn calving, but the cows tend to revert to the sexual season of their ancestors. One factor causing this change is probably the decreasing daylight hours in the autumn tending to favor not only conceptions but also multiple births (cf. Fig. 2) although a part is due to the length of the calving interval being about 13 months on an average. Fig. 2 shows the seasonal variation in twinning frequency in the same five breeds as referred to 4-6). in Fig. 1 and with the same classification on calving number. Multiple birth depends mainly on the number of eggs ovulated at a time when fertile spermatozoa are present in the oviducts, and therefore the diagrams relate the twinning frequency to the estimated month of conception. At the first calving, the Swedish breeds show a slight tendency to increased twinning frequency after autumn conceptions. For the Danish breeds and the German Schwarzbunt, this increase is quite pronounced. For these last mentioned breeds there is also a n increased twinning frequency after conceptions in May and June. At the second and following calvings all the five breeds show two very distinct peaks in the twinning frequency, one after spring and the other after autumn conceptions. The first peak occurs after conceptions in May (for SRB May and June), with calvings in February the following year, and the other corresponds to autumn conceptions and calving in the spring. In the diagram it may be noted that the maximum twinning rate of the cows in the Scandinavian countries occurs about one month earlier than in Niedersachsen. This supports the hypothesis that the calving as well as the twinning incidence in a district is to a certain extent regulated by the seasonal changes in length of daylight. In Table 4, the heifers are divided into two seasonal classes, autumn conceptions (September -November) and conceptions during the rest of the year (December-August). The twinning frequency is calculated for each class of the Swedish and Danish breeds. In each of the four breeds the seasonal difference in twinning frequency is highly significant. Table 5 presents similar data for the second and following calvings in the same four breeds. Since the cows show two seasonal twinning peaks, the data are divided into three classes according to conceptions in the spring, autumn, and the rest of the year. The differences in twinning rate between the three seasons are highly significant. The corresponding data for German Schwarzbunt in Niedersachsen are presented in Table 6, where classification is made on the first, second, third and fourth or more calvings. The seasonal differences in twinning rate are highly significant for all four parity groups. The spring maximum in twin conceptions may be explained as an effect of "flushing" when the cows are turned out on plentiful and lush pasture. It would be expected that individual animals react differently to "flushing" as well as to change in daylight hours, but there are probably, on an average, very slight, genetic differences between the groups of cows which are inseminated during the different months of the year. We are therefore inclined to believe that the spring maximum in twinning is an environmental effect, which could probably be produced at any time of the year, and that the autumn maximum is caused by the animals' reaction to the decreasing daylight. Effect af age of dam on twinning frequency Numerous investigations have shown that the age of the cow has a pronounced effect on the probability of her bearing twins (cf. HENDY and BOWMAN 1970). With one major exception, the ordinal number of parity would seem to be a satisfactory measure of the age of the cows in a population, and it is certainly the most convenient measure. The age at first calving varies within and between breeds, and therefore the influence of this variable was the subject of a special study. It would have been interesting to follow up the problem of the combined effect of age and parity for the second and third calvings but this could not be done on the available data. With increasing number of parities, the need for taking calving age into special consideration will gradually disappear. Fig. 3 presents in a general way the relation between age at first calving and the frequency of twinning in the two main Swedish breeds, SRB and SLB. The diagram is based on 202,468 SRB cows with an average age of 29.15 months at the first calving and 77,087 SLB cows with an average calving age of 29.78 months. The percentage distribution of the cows on fourmonths age-groups is shown below the diagram. Linear regressions have been fitted to the twinning frequencies. For the SLB, the fit is not very good, but the data for the highest age group indicated that some second calvers happened to be registered there. Therefore, no importance should be given to the deviation of this group average. The breed averages (7) The numbers of calving and twinning are estimated from pooled data for the seventh and following calvings stated above. Both regression coefficients, as well as the differences between the breed means for twinning frequency at the first calving are highly significant. Data on the relation of twinning frequency to age of dams, measured by number of parities are presented in Table 7 for the Swedish and German breeds, viz. Schwarzbunt (Black and White Lowland), Fleckvieh (German Simmental) and Braunvieh (German Brown cattle). In addition, pooled data are given for three Bavarian breeds where the number of cows was small. Only data for the first to the seventh calving inclusive are listed in the table. In the Swedish breeds, only a small number of cows had more than seven calvings, for the Bavarian breeds all data beyond the sixth calving were pooled at the data-processing, and classification on parity beyond this point was made only for Schwarzbunt in Niedersachsen. For the Bavarian breeds the number of cows at the seventh calving was estimated on the basis of the data from Niedersachsen . There was a discrepancy in the methods of sampling between the Swedish and German breeds which might have had an influence on the data on which the age curves are based (cf. Fig. 4). The German data represent a crosssection of all the tested cows in the year 1971 (Bavaria) or 1972 (Niedersachsen), whereas the Swedish data represent all cows which were culled in the period 1969-71. The history of these cows was followed retroactively until they entered the herds as calving heifers. The average twinning frequency was practically the same for each year from which the samples were taken, but the sampling method might have led to an exaggeration of the preponderance of young cows in the samples. Taking a cross-section of all the tested SRB and SLB cows during the period 1960-70, the first calvers amounted to 31 % of the total calvings in SRB and 34 % in SLB; the corresponding figures for the data presented in Table 7 are 36 and 40 % respectively. The average twinning incidence for parities 1-7 in the crosssection sample was for SRB 1.49 and for SLB 2.03 %, or very close to the figures given in Table 7. The culling rate among the young cows was apparently much higher in the Swedish than in the German breeds. The percentage first calvers in the total samples from the German breeds was for Fleckvieh 24 %, Braunvieh 23 % and for Schwarzbunt in Niedersachsen 26 %. In the same breeds 13 %, 17 % and 5 %, respectively, of the total number of calvings occurred at the seventh or later parities; the corresponding figure for the Swedish breeds was about one per cent. The figures show that when breeds are compared with regard to average twinning frequency it is necessary to correct for differences in the distribution of calvings on parities. This has been done in Table 7 with the SRB distribution as standard. With this correction, there is a significant difference in twinning frequency between the Swedish Friesians and the Schwarzbunt in Niedersachsen (0.41 0.038). The corresponding difference between Fleckvieh and Braunvieh is also significant (0.24 t 0.037) and both these breeds show significantly higher twinning frequencies than any of the other breeds listed in Table 7. The twinning frequency at the first calving is higher for Niedersachsen Schwarzbunt (0.83 %) than for the Swedish Friesians (0.63 %). This depends at least partly on the 2.2 months higher freshening age (32.0 contra 29.8 months). Fig. 4 presents a general picture of the relation between twinning frequency and age of the cows in the populations studied. Third degree polynomial curves have been fitted t o the observed frequencies. Comparing Fleckvieh and Braunvieh there is a marked difference in the age at first calving. At the time when the records were made, the average age at first calving was 29.3 months for Fleckvieh and 31.7 months for Braunvieh. This difference and the higher twinning frequency for Braunvieh at the 5th-7th calvings suggest that this breed is later maturing and more "persistent" at higher ages than the Fleckvieh. In the Fleckvieh the twinning rate rises more rapidly from the first to the third calving than for any of the other breeds. In this breed the seasonal distribution of the calvings is more uniform than for the Braunvieh but it is not known what in-fluence this difference has had on the age curves for twinning rate. whereas for the Niedersachsen Schwarzbunt the corresponding figure is only 2.28. The difference (0.57 %) is highly significant. This may be partly a genetical effect and partly a n effect of higher nutritional levels of the cows in Sweden and Denmark than in Niedersachsen as indicated by a 600-800 kg higher average milk yield of the tested cows. The possibility of genetic differences is supported by the fact that the present-day black and white cattle, particularly in Denmark but also in Sweden, are based mainly o n imports from Holland whereas the interchange of breeding stock between Germany and Holland has been rather small in recent times. However, the conclusion may be drawn that genetic differences exist between most of the breeds listed in Tables 7 and 9 with regard to twinning frequencies. The proportion of monozygous to dizygous In the literature, the frequency of MZ births is expressed in three different ways: corresponding frequencies of DZ twins in the same sample. Several human geneticists have expressed the opinion that the incidence of MZ twinning is "not appreciably influenced by the mother's age" (PENROSE 1963). BULMER (1970) has analysed human data from various countries and tried to separate the effect of age and parity on twinning rate. He concluded that the MZ twinning rate shows a "slight increase with maternal age, but fails to show any effect of parity". With regard to DZ twinning he found a clear increase in rate with parity but stated that this effect was smaller than that for age. ENDERS and STERN (1963) found, in studying large samples of the white and negro populations in the USA, a significant tendency for the frequency of MZ births to increase from the youngest group of mothers 15-19 years old to those approaching the climacterium, and this With regard to the present investigation, it would have been desirable to study the effect of maternal age not only on the total twinning rate, as in Fig. 3, but also the separate effects on the MZ and DZ rates within the first, and preferably also within the second calvings, but this could not be done without a costly rearrangement of the data for processing. In cattle, the correlation between maternal age and parity is so high, and the twinning rate so stabilized after the second calving, that further separation of the effect of maternal age and parity would probably be of no value (cf. JOHANSSON 1932). In Table 8, data are presented for the same breeds as in Table 7, showing the relation between age of dam and the estimated frequencies of MZ and DZ twin births. The total number of twin births in the breeds is somewhat higher in Table 8 than in Table 7, because the former table includes all registered calvings of the cows, even after the seventh calving. In Table 8, all data from the fifth and following calvings have been pooled because of rapidly decreasing numbers with increasing age of the cows, especially in the Swedish breeds. Furthermore, the change in twinning frequency after the fourth calving is very slight. There are two especially interesting features of the estimates of MZ twinning in Table 8. Firstly, when expressed in per cent of like-sexed twins, the MZ frequency is much higher for the first than for the following calvings. This is most Hereditas 78, 1974 marked for SRB and German Schwarzbunt and least for the Braunvieh. After the first calving there is no clear trend in the percentages with increasing age of the dam. Secondly, when the MZ twinning frequency is based on the total number of calvings, there is a more or less marked tendency for an increase up to the fourth calving. This is most pronounced in the Bavarian breeds, Fleckvieh and Braunvieh, and least in the Swedish breeds. Fig. 5 gives a visual presentation of the breed differences in the relation between maternal age and the MZ and DZ twinning rates. The two Bavarian breeds are very much alike in the average rates of MZ and DZ twinning and their relation to maternal age. Therefore the data for these two breeds were pooled and averages calculated on which their age curves are based. The two Swedish breeds, SRB and SLB, differ significantly in average DZ rate (0.52 f 0.036) but they are rather similar in MZ twinning rate. For SLB the estimated numbers of MZ pairs in the various parities are very small, and the group averages behave therefore rather irregularly. For this reason the MZ data for SRB and SLB were pooled, but the DZ data were kept separate. The third breed-group is the Niedersachsen Schwarzbunt which is about intermediate to the other two groups in MZ twinning rate. The group differences in MZ frequency are relatively small at the first calving but later on there is a rapid rise in the Bavarian breeds up to the fourth calving, followed by a decline at the fifth and sixth calvings. The difference between the MZ frequency, in the first and fourth calvings is highly significant (0.392 k 0.037), and thefollowing decline to the sixth calving is also highly significant (0.248 f 0.029). In the Niedersachsen Schwarzbunt the trend is similar but much less pronounced. The rise in MZ frequency, from the first to the fourth calving is significant (0.130f 0.049), but the following decline to the fifth calving is barely significant (0.088 f 0.046). On an average for the two Swedish breeds, the MZ twinning rate is significantly lower at the first three than at the following four calvings (0.076 f 0.026). A rather peculiar phenomenon is, that in all three breed groups the MZ twinning incidence attains a relatively high level at the fourth calving, declines at the fifth and sixth calvings and then rises again for the older cows (7 I calvings). For the Swedish breeds. the total number of the seventh and following calvings was 7,304 with an estimated number of 59 MZ births (= 0.81 %). The relation between maternal age and DZ frequency is nearly the same for SRB and the Niedersachsen Schwarzbunt as shown in Fig. 5. The age curves for both breeds are plateauing after the fourth calving. The comparison between SLB and the Bavarian breeds is interesting. At the first three calvings the DZ frequency is higher for the Bavarian breeds (B + F) but then the age curves cross one another, and at the 5th-6th parities the DZ rate is higher for SLB than for F +B. The later downward and upward trends in the age curves are not significant. Some additional data on the relative incidence of MZ and DZ twinning are presented in Table 9 for the Danish breeds and Swiss Simmental, where classification was made only on the first and the following parities. The incidences of MZ twinning, calculated in per cent of total births, are surprisingly low for the Danish Red and Black and White breeds but they are rather high for the Danish Jersey, even compared with breeds of large body size. The DZ rate is very low in the Jerseys, on an average for the total number of calvings 0.89 compared with 0.242 % MZ twins. In the Swiss Sirnmental, the DZ and MZ frequencies are both high. The conclusion may be drawn that not only the DZ but also the MZ twinning rate depends on the age of the mother. In the Fleckvieh and Braunvieh, the relative rate of increase from the first to the fourth calving is just as pronounced as the increase in DZ twinning rate, but in the Swedish breeds it is very slight. Apparently, there are genetic differences between cattle breeds in the rate of increase in both MZ and DZ twinning with increasing maternal age. On the whole, however, the MZ twinning rate tends to be more stable throughout the life of the cows than that of DZ twinning. Incidences of triplet births The records on triplet births in the Swedish breeds are incomplete, but records which seem to be fairly complete are available for Danish and German breeds and for Swiss Simmental. Multiple births of higher order than triplet have occurred but they have not been regularly registered, or in some cases registered as triplet births. On the basis of human data HELLIN ( formulated the rule that when the ratio of twin births to the total number of births is 1 : X, triplet births occur according to the ratio 1 : X2, quadruplets 1 : X3, etc. Some later investigators have found fairly good agreement between observed ratios and HELLIN'S rule. However, more sophisticated methods have also been used (cf. BULMER 1970) where the biologically different types of zygosity in sets of triplets, quadruplets, etc. have been considered. It is well known from human genetics that the multiple sets may be all MZ, all DZ, or any possible combination of zygosities. In cattle, several cases of monozygous triplets and at least one case of monozygous quadruplets (DONALD et al. 1951) have been reported. In recent years, breeding stock of three beef breeds have been imported to Sweden for crossbreeding purposes, viz. Hereford and Aberdeen Angus from Great Britain and Charolais from France. The number of purebred Aberdeen Angus cattle in Sweden is very small, and our data from this source comprise only 1,210 calvings with 18 twin births (1.49 f 0.348 %) which is too limited to be a satisfactory random sample o^ the b:eed. In Table 11, the twinning frequency in the Swedish Charolais is compared with corresponding records from France, and the Swedish Herefords Hereditas 78,1974 are compared with data from the Wyoming Hereford Ranch. The distribution of calvings on parities is expressed as a percentage of the total calvings in order to facilitate the comparisons. The figures show that for both breeds the Swedish cows are on an average younger, than the cows in the comparable population samples. For the same ordinal number of parity, the Swedish Charolais cows tend to show a somewhat higher twinning frequency than the French Charolais, and if the Swedish average is corrected to the same distribution on parities as the French material, the Swedish average would be 3.65 % twinning, compared to 3.19 % for the French Charolais. The twinning frequencies for the Swedish Hereford are somewhat higher throughout than the corresponding figures for the Wyoming herd, but the difference between the averages is not significant. It might be expected that a herd under ranch conditions would show a relatively low rate of twinning. The difference between the Swedish Charolais and the Swedish Herefords, kept under the same environmental conditions, is highly significant in spite of the small numbers (2.15 f 0.455). Monozygous twinning occurs apparently with about the same relative incidence in the beef breeds as in the dairy breeds. By pooling the Swedish and the Wyoming data for the Herefords we find the following sex combinations of the twin pairs: 6266+80@+89&?. It For the first calving the percentage was almost twice as high, or 43.6 %, thus agreeing with the situation in the dairy breeds (Table 8) that the relative incidence of MZ twinning is higher at the first than at the following calvings. Inheritance of the twinning tendency The inheritance of twinning has been extensively studied by human geneticists from family records or population samples. It has been shown in the U.S.A. that the frequency of twinning is higher for the black than for the white population. It is particularly low in Japan, and in Europe it is higher in the Fenno-Scandinavian than in the Mediterranian countries. Many researchers have considered it likely that the tendency to bear twins is a recessive trait with simple Mendelian inheritance (cf. PENROSE 1963). However, STERN (1960) writes "If the genetic basis of twinning is a simple one, it must have a very low penetrance ". DAHLBERG (1952) found in a study of 2,222 births that after the first twin birth, repetitions of twinning were on average only 5.46 f 0.49 %. In previous sections it was found that different cattle breeds sharing the same geographical area may show highly significant differences in twinning rate, whereas closely related breeds, existing in different areas, usually show rather similar twinning rates (Tables 7, 9 and 11). This leads to the assumption that the variation between individuals within the same breed is, at least to some extent, of genetic origin. In accordance with the majority of present-day opinion, we assume that predisposition for twinning is a polygenic character with a discontinuous manifestation on the observed scale. The analysis of such characters can be performed on the observed (binomial) scale or according to the assumption of an underlying continuous variation. The problem has been discussed as regards out that heritability on the observed binomial scale (p-scale) will be influenced by the frequency of the character, due to the dependency of the variance on the mean, and by the coarseness of grouping which of necessity decreases the correlation between observation and genotype. They suggest that heritability on the two scales can be approximately interconverted by the following relation: where h2p = heritability on the observed scale h2, = refers to the continuous underlying scale q =frequency of the trait in the population, and p = 1 -q and z = ordinate at the threshold point, which separates the two classes, assuming normal distribution of the twinning liability Falconer suggested a simple and elegant method to estimate heritability of the underlying variable for threshold characters. His model can be used for twinning which on the observed scale is an all-or-none character. Underlying this binomial distribution is the liability for twinning which not only includes the "individual's innate tendency" to give birth to twins but also "the whole combination of external circumstances" influencing the rate of twinning, such as age, season of conception, etc. All individuals where the liability exeeds the threshold will exhibit twinning. The position of the mean liability of a population (XP) can be expressed relative to this threshold, as can the difference between the mean IiabiIity of twinbearing individuals and the population mean (=z/q=a). If differences in liability are partly genetic, relatives of twin-bearing cows should have a higher mean liability (XR) which will be closer to the threshold. Falconer suggests estimating heritability by the increase in mean liability of relatives of twin-bearers over the population mean in proportion to the mean superiority of other all-or-none or quasicontinuous characters liability of the latter, i.e. h2X -X P -X R , where by, among others, LUSH, LAMOREUX and HAZEL r . a (1948), and FALCONER (1965, 1967). Since we use r = coefficient of relationship between twinboth approaches, the problems involved may be bearers and relatives. This approach can also be briefly discussed. used for estimating repeatability and the im- A. Repetition and repeatability A suitable starting point in studying the possible effect of heredity on twinning would seem to be the repetition of twinning after the first twin birth. Repetition refers to the twin-bearers only, whereas repeatability refers to the whole population of twinning and nontwinning cows (cf. BOWMAN and HENDY 1970). The rate of repetition shows simply the proneness of individual cows to repeat twinning at the following calvings regardless of the underlying causes. An attempt to explain the inheritance of the twinning tendency on a monofactorial basis would be justified only if the repetition rate was relatively high. Table 12 shows the results obtained in an analysis of data from the two Swedish breeds SRB and SLB with regard to the repetition of twinning in different age groups and after the first and second twin births. It has been assumed that early twinning, at the first or second calving, is a stronger indication of genetic predisposition than when the first twins are born later in life, and therefore the data are classified according to the parity when the first twinning occurred. No significant differences appear between repetition rates at various parities when tested by x2. However, in SRB 32% and 9 % of the twin pairs born at first parity and later parities, respectively, were probably monozygotic and the corresponding figures for SLB are 22 and 5 %. According to BULMER (1970) the tendency to repeat twinning in man is limited to DZ births, and the same probably applies to cattle. If the rate of repetition is estimated relative to dizygous twinning only, the repetition would be 9.4 and 5.8 % respectively after heifer and later twinning for the SRB, the difference being significant (x2 = 4.1). The corresponding figures for the SLB are 11.5 and 10 %. Therefore it appears that the suggestion that the repetition of DZ twinning is higher for heifers than for cows is a valid one. According to Table 12 there is a breed difference in the rate of repetitions after the first twin birth, and this difference is highly significant. The difference between the repetitions after one and two twin births is significant for SRB only (P < 0.05). For comparison, results from the earlier study of the same breeds (JOHANSSON 1932) are quoted in Table 12. The repeat figures were higher in this study than those obtained from the present data which probably has two main causes, viz. the earlier first calving and the shorter life of the cows in recent years than in the 1920's. Table 13 presents an analysis of variance of the population data for the Swedish Red and White cattle employing the same type of model as that used by BOWMAN and HENDY (1970). The repeatability of twinning (rp) in the population was estimated and since the number of calvings is fairly high, especially in the lower age groups, the estimates show a continuous series of increasing values from two to seven completed calvings in the groups. This increase would be expected because of higher twinning frequencies in older cows. Falconer's method (FALCONER 1965(FALCONER , 1967 involves a slight downward bias of the estimates. When corrections were computed they were found to be almost insignificant and therefore only the original values are reported. Repeatability of liability of twinning (rx) can be estimated from repetition rates: SRB As is apparent from these figures the repeatability of twinning liability of the two breeds is significantly different, albeit the difference is relatively smaller than that of the repetition rates. When converted to the observed scale, the repeatability is a little larger than that of Table 13 but these latter values are probably too small, as evidenced by the fact that they are below the similarly estimated heritability figures (p. 223). On the other hand, since repetition rate was compared with the population frequency of twinning, the intraherd repeatability may have been overestimated if real herd differences in twinning frequencies exist. However, this could not have been very great as will become evident in the second paragraph to follow. It may be stated that cows show differences in their predisposition to twinning and that these differences become more evident when cows with repeated records of twinning are compared with average animals. Breeds In the Bavarian data the twinning frequency of daughters was compared to that of their herd mates (cf. Table 17). The differences between the latter and the population average twinning frequency can be used for investigating the existence of real herd differences in twinning liability: The b values indicate to what extent herd differences in the liability of cows are repeatable. It can be concluded that real herd differences d o exist, probably mainly due to differences in feeding and management. BAR-ANAN (1973) B. Heritability A preliminary analysis was made of the records of A I bulls with large numbers of offspring. Regarding twinning averages of bull-progeny groups calculated from such data, herd differences are unimportant because the daughters of each bull are spread over a large number of herds, but the influence of age differences should be considered. The daughters of each bull were divided into two groups, viz. heifers and cows, and their ratio was used to divide the bulls into 5 subgroups (Table 14). A high ratio was assumed to indicate that the daughters that had reached cow stage (2 I parities) were relatively young. The twinning averages for the cow groups indicate that at least part of the differences in age distribution of progeny groups was eliminated by this grouping. A X2-test of the differences in twinning frequency between the 5 cow-groups turned out to be highly significant (xz = 81.35), while it failed to reach significance for the heifer groups ( x Z = 6.42). Table 14 is arranged so that daughters of the same bull are entered on the same row, but heifers and cows separately. Since the twinning frequency at the first calving is fairly low, the difference between sires with regard to twinning frequency at the first calving of their daughters surpasses the conventional level of significance only twice in each breed. However for later parities, the differences between bulls are highly significant in each one of the five groups. Using the X2-method (ROBERTSON and LERNER 1949) the following half-sib correlations (within groups) and heritability estimates were obtained: analysed. The heritability was estimated at 0.0178 f 0.0014, i.e. only slightly different from the preliminary estimate. These total data were not grouped according to heiferlcow ratio which may be the cause of the difference. The distribution of the twinning frequencies of the progeny groups of the 101 SRB bulls and 34 SLB bulls, the latter from table 14, are shown in Fig. 6. The average twinning frequency for 226,833 calvings of cow-daughters of 101 SRB bulls is 2.21 %. However this figure is reduced to 1.88 % when each bull is credited for exactly 1,OOO daughters and the corresponding number of twin births. The corresponding average for the cowdaughters of the 34 SLB bulls is 2.51 %. The distribution of the average twinning frequencies in the progeny groups shows a pronounced skewness in both breeds. BAR-ANAN and BOWMAN (1974) observed a similar skewness in the distribution of twinning frequences in progeny groups from Israeli-Friesian bulls, and they suggest that a major gene for twinning might be involved. However, the skewness may by expected if we assume that the genetic distribution on the underlying scale is itself normal. In another analysis of partly the same data as in Table 14, repeated progeny tests of A I bulls were made on the twinning frequency of their daughters. Two random samples of daughters as heifers and cows were used for each bull. For heifers the sample size was 500 + 500 daughters, but 1 ,OOO + 1 ,OOO calvings were used for the cows. In order to exclude age differences, records were allotted alternatively in chronological order to the two subgroups. With regard to the heifers the numbers of daughters and calvings are identical, but cows may in rare cases appear in both samples, or twice in the same progeny sample. The correlations were computed between twinning frequencies of the corresponding subgroups (Table 15) Practically the same value of the half-sib correlation was obtained for SRB as from the data in Table 14. The SLB numbers (8 groups) are too small to attract any attention to the higher value. Many more bulls were used for the corresponding estimation from Table 14. Four earlier reports on the heritability of multiple births in cattle may be mentioned, published by MAIJALA (1964), , GAILLARD (1973) To summarize, it can be said that the heritability of twinning frequency of cows as observed is real but small, and that the heritability for heifers is even smaller. This difference may be explained by the very low dizygous twinning rate in heifers. For an estimation of twinning heritability on the continuous scale, we have two sets of data, a limited sample of cows from SRB and SLB and a fairly large amount of data from Bavarian Fleckvieh and Braunvieh. The Swedish data were manually selected from the milk-record files, and therefore the breed samples are rather small. Cows with one or several twin births (propositi) were picked at random and then their daughters (or dams) were located for comparison with the twinning records in the general population. In order to eliminate the effect of age at calving on the twinning frequency, only records for the 3rd-5th parities were compared. However, the propositi were used whenever they had born twins in the 1st-5th parities. For the SLB, where the largest sample was available, a special comparison was made for daughters of dams with two or more twin births. In the other cases only one twin birth per dam, and daughter was considered. Twinning frequencies of daughters and dams of propositi were computed, and the results are presented in Table 16. As expected, the heritability values estimated according to Falconer's method are much higher than those computed from the observed p-scale. Estimates for SRB are somewhat higher than those for SLB which is the reverse of that found for estimates on the observed scale. When the ha estimates are transformed to the binomial scale, the results are very close to those estimated directly from the observed twinning frequencies of cow progeny groups in Tables 14 and 15. The transformed hZp values are roughly equal to the similarly estimated repeatability of twinning in SRB but smaller than repeatability in SLB (cf. p. 221). Herd differences were not taken into account in any of these calculations. For the Bavarian Fleckvieh and Braunvieh a greater amount of data were available. Both twinning dams and their daughters could be grouped according to parity and the daughters could be compared with unrelated, contemporary herdmates in order to exclude possible effects of herd environment on the twinning frequency. The results are shown in Table 17. The number of first parity daughters from first parity dams was rather small in both breeds and their relatively high twinning frequency does not deviate significantly from the average of the other first calving groups of daughters. Since the twinning rate of the daughters seemed to be rather independent on how early their dams started twinning, averages were calculated for each parity group of daughters from twinning dams of all ages. The twinning averages for the total population samples are stated in parentheses below the corresponding average for the contemporaries. For Fleckvieh where the largest numbers were available, the twinning frequency of the daughters of dams with at least two twin births was also calculated for each one of the parity groups. It is interesting to note that the twinning frequency among the daughters of dams with two or several twin births is consistently higher than for daughters of dams with only one registered twin birth. This is reflected in higher h2, values. It is in good agreement with the results presented in Table 12 showing higher repetition of twinning at calvings succeeding second twin births than when the cows had born twins once only. The heritability values of single twin births vary between pairty and breed groups but no obvious pattern can be discerned. Transformation of the h2s values yields heritabilities fairly close to those reported by GAILLARD (1972) from Simmental progeny groups, and to the Swedish results discussed above. Transformed hZp values of "double twinning" are about twice those of single twinning but still quite small in absolute terms. The h2, estimates from the Swedish Friesians (SLB) and the Bavarian breeds lie in the same range of values. The SRB estimates are slightly higher but the samples from this breed are rather small and the difference between SRB and SLB is not statistically significant. Moreover the large h2, values for SRB are not reflected by large repeatability values (Table 12). The transformation reduces the various hZX values to estimates which are fairly close to the directly computed binomial heritabilities. No bias in any direction can be discerned. In contrast, The comparable twinning frequency in the general populations is stated within parentheses. h2,= heritability of the normal scale; h2,= transformed value on the N 2 binomial scale DEMPSTER and LERNER (1950) found higher heritabilities for egg production from direct estimation on the binomial scale than when the regular estimate was transformed by multiplication with -. However, in view of the standard errors, they felt that the agreement was satisfactory. Also VAN VLECK (1972) found from computer simulation experiments that heritabilities estimated directly from the binomial scale were larger than those obtained by transformation. As indicated, we find the agreement satisfactory and without apparent bias. Falconer's method therefore appears to be a convenient and efficient way of estimating heritabilities for rare traits from large volumes of data. It directly estimates the liability for the trait which, after all, is responsible for the observed correlations between relatives. Of course, the latter are relevant for animal improvement but as our results indicate, they can be estimated sufficiently well from knowledge of the heritability on the underlying continuous variable. As will be shown later, h2s can also be used for estimating genetic gain from select ion. C. Genetic correlations Twin births in different parities are composed of varying fractions of MZ and DZ twins. As has been indicated above, it appears, that the frequency of MZ twins is more stable between breeds and parities than that of DZ ones, and that only the frequency of the latter shows appreciable heritable variation. The DZ twin frequency varies greatly with age. Therefore, the question may be raised, whether twinning at the different parities is genetically identical, i.e. whether the genetic correlations are 1. The large volume of Bavarian data permitted estimation of heritability of and genetic correlations between twinning liability in lst, 2nd and later parities. Regression coefficients were computed for daughter-herdmate differences in liability a t different parities on dam superiority for twinning liability in each parity group ( I , 2 and later). The coefficients were not significantly different for the two breeds and therefore the estimates were combined. The genetic correlations (rc;) between parities and the heritability (h2) within parities were as follows: The heritability estimates vary, but the genetic correlations between twinning liability in different parities appear to be 1. Obviously this refers t o DZ twinning liability only. This seems reasonable if one assumes that MZ twinning liability has no, or very slight, genetic basis, and that it has no genetic connection with the predisposition for DZ twinning, which assumption agrees with most observations reported in the literature, as indicated above. D . Paternal influence on frequency and zygosity of twins A priori it seems unlikely that the sire of the calves has any influence o n the DZ twinning rate, which primarily is determined by the number of eggs ovulated in connection with the heat period when insemination takes place, but it is quite possible that the sperm, as well as the egg, conveys genes to the zygote which makes it split at a n early stage of development, thus producing monozygous twins, as suggested by DAHLBERC (1926). With the exception of the Jersey breed and the first parity of the other breeds listed in Tables 8 and 9, the MZ twins constitute only a small part of the total number of twins produced, and they have therefore only a slight effect on the total twinning rate. However, for the sake of comparison we tested the variation in twinning rate after parturitions where the bulls were sires of the calves. This was done by the same type of progeny test as used in Table 15. The results are shown in Table 18. In both heifer groups and also in the SLB COWgroup the correlation between twinning rates of the duplicate samples are negative but not significant, but in the SRB cow-group the correlation is positive and significant at the P < 0.01 level. Hcrrdiras 78, I974 We are not inclined to consider this as an evidence that some sires are predisposed to produce more twins than others. The deviation from the total sire average may be due to some unknown causes. Another analysis was made of the distribution of like-sexed and unlike-sexed twins on 13 SRB sires that had each produced at least 100 twin pairs, with a range of 101 to 275 per bull. Of the total number of 2,138 twin pairs, 1,119 were like-sexed and 1,019 unlikesexed. The estimated number of MZ pairs would therefore be 1 0 0 or 8.94 % of the like-sexed pairs. The observed numbers of like-sexed pairs from the individual bulls were fairly close to the expected numbers. The deviations from the average for the 13 bulls were not significant (xz = 11.54 and P -0.50). The results did not indicate that the sire has any influence on the production of MZ twins. This is in agreement with results obtained from analyses of human data, reviewed by BULMER (1970). Possibilities of selection Twinning in cattle has some disadvantages as discussed in recent reviews by HENDY and BOWMAN (1970) and by HOLZ, HARING and SMIDT (1970). The number of premature births increases, and the perinatal mortality is about four times higher than for single-born calves. The length of calving interval after twinning increases due to frequent complications during and after parturition. Furthermore, about 90 % of all female calves born co-twins with bulls are sterile because of intersexuality. Therefore, some natural and possibly also some artificial selection operates against twinning. Table 19 bears this out. Culling rates are higher after twinning than in the general population and elimination of cows after repeated twinning is especially high. The increase in culling may result from reproductive disturbances and/or conscious selection by the breeders. SYRSTAD (1973) found that in Norway the culling rate was about 5 % higher after twinning than after single births. Thus, it is arguable whether selection for higher twinning frequency in dairy breeds would be profitable. However, the disadvantages are not so great that selection against twinning would be likely to justify the efforts, as was pointed out by ERB et al. (1960). The situation is different in the dual purpose beef-milk breeds and in some specialized beef breeds. In spite of the larger losses among twin calves, more calves are produced than from single births. BAR- ANAN and BOWMAN (1974) have estimated that from 100 twin births some 160-170 calves can be raised compared to about 95 from 1 0 0 single births. The Hereford and Aberdeen-Angus breeds are characterized by low twinning frequencies, and the cows give so little milk that in some cases it is hardly sufficient for one suckling calf. MECH-LING and CARTER (1964) have reported on an attempt by a private breeder to raise the twinning frequency in an Aberdeen-Angus herd by selection. Only twin bulls were used as sires in the herd, and the females used for breeding were born as twins or were daughters of twinning cows. After some 15-20 years the average twinning frequency in the herd was 1.71 % in 585 calvings. If any real progress had been made, it was certainly very slight. Selection pressure and/or accuracy were too weak. In Australia, experiments have been carried out at two research stations on the possibility of raising the rate of multiple births of Merino sheep by selection, and TURNER (1966, 1968) has reported good results. The selection was carried out in the same environment over a period of 10-12 years, in one line for low and in one line for high rate of multiple births when the ewes had reached three years of age. At the conclusion of the experiment the difference between adult ewes of the two lines was at one of the stations 35 lambs per 100 ewes mated. Selection on the number of lambs when the ewes were two years old had little effect. A selection procedure with the purpose of increasing the twinning frequency in cattle should preferably start with a dual purpose beef-milk breed with a high initial twinning frequency and a milk yield which is high enough for nursing two calves at the same time, for example the various Fleckvieh strains. Among the specialized beef breeds we have studied, the Charolais shows the highest twinning frequency but Fleckvieh and Simmental are superior in milk yield. Since the heritability values are very small, reasonable accuracy can only be achieved when selection is based upon information from many observations. This leads immediately to progeny testing of bulls on large numbers of daughters with mature calving records. Accepting 2 % as the heritability of twinning in adult cows would imply a repeatability of more than 0.8 for bulls with 1,OOO daughers. On the female side the gain in accuracy is limited even when using repeated records. However, selection pressure will be strong when twin-bearing cows are selected or selection is restricted to cows with repeated records of twinning. Let us assume that in a project aimed at increasing twinning frequency 20 % of the bulls with the highest twinning rate in their daughters are selected, e.g. among the 101 SRB bulls, the selection differential would be (3.64 -2.21 %) = = 1.43 7;. Assuming 1,000 daughters per bull and h2 = 0.02, the repeatability of the progeny test will be 83 % so that future daughters of the same bulls should have increased the adult twinning frequency by 1.19 % to 3.40 %. Cows with adult records involving two or more twin births would have a liability which is 2.38 standard deviations above the mean. A heritability of 2 % on the binomial scale implies a heritability of liability of 0.156 when the population incidence is 2.21 %. Progeny from such cows should be 0.1 86 standard deviations above the mean liability, which should raise their twinning rate to 3.73 %, a gain of 1.52 %. Therefore, in a selection program which involves mating of the 20 % best progeny-tested sires to cows with two or more twin records, the twinning rate should improve by more than 2 % per generation (2.71 % in our example). The first aim of such a program would be the production of young bulls for future breeding. It is not our intention to draw any detailed breeding plans but simply to show that an intense selection for increased twinning rate can be expected to yield quite satisfactory response. -It seems likely that by selection through many generations it would also be possible to produce cows that are better adapted to multiple births than those existing today. Discussion Several investigations indicate that multiple ovulation in the cow is more common than multiple birth . KIDDER^^ al. (1952) and L A B H S E T W A R (~~~~) reported 6.1 % multiple ovulations from 900 parities with 3,549 ovulations. Conception rate after single ovulation was 57.5 % but only 26.8 % after multiple ovulation. Out of 12 diagnosed pregnancies after multiple ovulations three involved twins (1952). Cystic ovaries were diagnosed in 62 parities involving 478 ovulations. In cystic periods multiple ovulations were about three times higher than in normal heat periods (1963). SETTERGREN (unpubl.) examined genital organs from slaughterhouse material of US Holsteins and SRB. In the Holstein material two corpora lutea (c. 1.) were found in 58 (6.4 %) and three in one of 901 organs with no detectable pregnancy. Fourty of the 58 double c.1. were on the same ovary and of the remaining 18 one on each side. Out of 113 pregnant uteri seven contained twins and in five of these cases one c.1. was on each ovary. The SRB material comprised 198 nonpregnant uteri where in three cases two c.1. were on the same ovary and in four cases one on each side. Four of 88 pregnant uteri carried twins. In one case the two c.1. were on one ovary and both fetuses in the corresponding horn, in two cases the two c.1. and the two fetuses were distributed bilaterally and in the remaining case only one c.1. was present with like-sexed twins in the corresponding horn. SETTERGREN examined Graafian follicles by serial sections of about 50 ovaries and found only single ova in each follicle. His opinions was that intrauterine migration of fertilized eggs or blastocysts is rare. ROWSON (1973) found only two twin pregnancies out of 16 when two eggs were transferred into one uterine horn. Transfer of one egg into each horn resulted in 73 % twin pregnancies. Low twinning rate upon egg transfer into one horn was attributed to lack of uterine migration and death of one embryo due to competition, usually at about 50 days post conception. ROWSON, LAWSON and MOOR (1971) and The Wisconsin investigations and a report by HENRICSON (1956) found in 149 SRB herds in Middle Sweden the frequency of cysts to increase from a low value in heifers to a maximum in 4-5 year old cows. The frequency had its seasonal peak November-February and its minimum May-September. This cycle is rather different from seasonal variations in twin conception (cf. Fig. 2) but it correlates with variation in non-return rate. The reproductive processes of the cow represents a complex series of inter-related events, the sequence of which is hormonally regulated. In the majority of cases only one ovulation occurs at one heat period and smaller follicles degenerate. The regulation of ovulation is not perfect, however, and in some cases two or more follicles ovulate at the same time. The hormonal equilibrium necessary for single ovulation is apparently more labile in some cows than in others and this may lead to repeated multiple ovulations in certain animals. Their ovulatory mechanism is less well canalized, to use a genetic term. This may be considered quite normal but the distance may be short to pathological conditions where no ovulation occurs and ovarian cysts are formed. ERB and MORRISON (1959) found a 3-5 fold increase in frequency of retained placentas after twin births compared to single births. Also, ovarian cysts and persistent c.1. became more frequent, obviously a result of hormonal imbalance. Thus twinning may sometimes be a cause for the immediate repetition of twinning, MZ twinning is a biologically different phenomenon. Twins arise from one fertilized egg. BULMER (1970) concludes, from a survey of statistics, that "propensity to MZ twinning is the same in all women" but that "propensity to have DZ twins varies from woman to woman". The validity for cattle within a breed, of the first part of this statement may be an open question although it agrees with our own findings (cf. p. 229). BULMER further concludes that there are no genetic differences between human races for MZ twinning frequency. ENDERS and STERN (1948), however, found the increase in MZ twinning frequency with maternal age in US negroes and whites to be significantly different. BULMER'S statement certainly does not apply to cattle where highly significant breed differences have been found (cf. Tables 8 and 9 and Fig. 5) and breeds differ also in the age-related increase of MZ twinning rate (Fig. 5) . We conclude from our investigations that DZ twinning frequency is a polygenic character with a threshold. The rather complex physiological mechanisms which determine single or double ovulation, and the complexity of the further course of events such as migration of ova, success of implantation, competition etc. would support this contention and lend plausibility to the genetic model we have used. Whether a single locus in a given genetic situation can play a major role may be left as an open question. Summary Cow-testing records from 10 European dairy breeds and two specialized beef breeds were analysed. The main results may be summarized as follows. ( I ) The sex-ratio for abortion and perinatal mortality for single-born and twin-born calves is 60-70 % males. The mortality of twin-born calves is 3-4 times higher than for single-born ones (Tables 1 and 2). (2) Season of conception has a pronounced influence on the frequency of twinning. Primipara reach the highest frequency after conceptions in October, whereas cows (2 I calv-ings) show two yearly maxima, viz. after May and autumn conceptions (Fig. 2). (3) Twinning frequency is low at the first calving but gradually increases up to the 4th-5th parities and flattens out later on (Fig. 4). (4) The frequency of monozygous twinning, expressed in percent of total calvings, varies between breeds and parities. In the Bavarian Fleckvieh and Braunvieh there is a marked increase up to the fourth parity with a decrease later on. For the Swedish breeds the frequency is approximately constant for the first three parities, and the later increase is relatively small (Fig. 5). On the whole, the frequency of MZ twinning is less agedependent than D Z twinning. (5) The repetition of twinning after the first twin birth was found to be 6.07 % for Swedish Red and White cattle (SRB) and 9.72 % for the Swedish Friesians (SLB). After the second twin birth the corresponding figures were 10.75 and 15.30 % respectively ( Table 12). The repeatability of twinning was estimated for the SRB population only. It rose from 0.0009 for completed first and second calvings to 0.0154 with seven completed calvings (Table 13). (6) The variations in twinning rate are probably dependent on many genes with complicated interactions between the genotype and a multitude of environmental influences, especially nutritional and seasonal variations. Phenotypically, twinning is an "all-or-none'' trait, but it is likely that the underlying liability to bear twins has an approximately normal distribution. (7) Highly significant differences were found between A I bulls with regard to the twinning frequency of their daughters ( Table 14). The correlation between progeny subgroups from the same sires, each subgroup comprising 1 ,OOO cow-daughters, was approximately 0.8 (Table 15). When measured on the observed phenotypical scale, the heritability of twinning frequency was estimated to be about 2%, based on one twin birth per cow. When measured on the continuous x-scale the corresponding estimate was about 10 % for one and 20 % for two twin births per cow (Table 17). (8) Paternal influences upon total twinning frequency are probably unimportant, and no evidence was found for the suggestion that sires influence the rate of MZ twinning. (9) Intensive selection for increased twinning rate could be expected to yield a gain of at FALCONER, D. S. 1965. The inheritance of liability to cer-

v3-fos

2020-12-10T09:04:22.402Z

1974-07-01T00:00:00.000Z

237229022

s2

Influence of Viral Envelope on Newcastle Disease Virus Infection The adsorption characteristics of Newcastle disease virus (NDV) propagated in chicken cells (NDV-C) and in human cells (NDV-H) were examined. Adsorption experiments performed at different temperatures indicated that virus propagated in a particular cell infected that cell type more readily than did virus propagated in a different host. For example, NDV-C was more efficient in initiating infection of chicken cells at 22 C than was NDV-H; the reverse was true when human cells were employed. The results indicate that infection of susceptible cells by NDV is influenced by the host cell in which the virus was propagated. The data also suggest that NDV may be useful in studies on homologous and heterologous membrane-membrane interactions. Newcastle disease virus (NDV), a paramyxovirus, possesses an inner nucleocapsid surrounded by a lipoprotein envelope derived, in part, from the host cell. That NDV and related myxoviruses acquire structural material from the host cells in which they are propagated is well established from serological studies (11,21), electron microscope observations (17), and chemical analyses of similarities in host membrane and viral envelope material (5,8,9,10). This intimate association of host membrane components with virus has been suggested as an explanation for various changes (e.g., plaque morphology, density) observed in NDV when grown in different host cells (2,3,22). We have shown that host components in the NDV envelope influence the induction of early polykaryocytosis, a phenomenon related to the initial stages of NDV infection (24). In the present study, an attempt was made to examine the early interaction of NDV propagated in chicken and human cells with their respective host cells, and to determine whether the previous virus host influenced the infectious process. MATERIALS AND METHODS Cells. HeLa cells, obtained from W. A. Cassel, were grown in Eagle minimal essential medium (MEM) containing 5% calf serum. Chicken embryo fibroblasts (CEF) were prepared from 10-day-old embryos by conventional procedures (18); MEM was used as growth medium. For the experiments and assays to be described, secondary cultures of CEF were used. Viruses. The OS strain of NDV (1) was used. The original virus suspension was obtained as an allantoic fluid suspension, and from this two virus stocks were prepared as follows. (i) Ten-day embryonated eggs were inoculated with 200 to 500 hemadsorption focal units (HFU) per egg. Infected eggs were incubated at 37 C for 40 to 44 h prior to collection of allantoic fluid. Virus so prepared was designated NDV-C. (ii) HeLa cells were infected with virus at an input multiplicity of 1 HFU/cell. Virus harvested after 24 h at 37 C was designated NDV-H. The ratio of infectivity to hemagglutinating units was the same for both virus stocks. The density of NDV-C and NDV-H was determined by equilibrium sedimentation in CsCl and found to be 1.18 and 1.25 g/ml, respectively (24). Virus suspensions utilized in certain experiments were dialyzed against several changes of distilled water at 4 C for 24 h. This procedure effectively removed most of the ions present in the original suspension as indicated in Results. There was no decrease in virus infectivity after dialysis. Virus purification. Virus was precipitated from fluids by the addition of solid polyethylene glycol (Carbowax 6000) to a final concentration of 6% (wt/vol) (14). The virus was suspended in water and banded in a preformed gradient of 2 to 45% potassium tartrate (13). The gradient was centrifuged in an SW65 rotor in a model L2-65B Spinco ultracentrifuge at 42,500 rpm for 105 min at 4 C. Virus was collected from a sharp band and dialyzed against several changes of phosphate-buffered saline (PBS, pH 7.2) prior to use. This procedure was used for both NDV-C and NDV-H for some experiments (see Results). Infectivity assays. NDV suspensions were assayed on either HeLa or CEF cells as required. Virus titers were determined by a hemadsorption focus assay (1). For the assay on HeLa cells, a fluid overlay was utilized and plates were incubated at 37 C for 24 h 26 before the number of hemadsorption foci were counted. A solid overlay consisting of equal parts of 1.8% agar (Difco) and twice-concentrated MEM with 5% calf serum (final concentration in overlay mixture) was used in assays on CEF. Titrations on CEF were incubated at 37 C for 48 h prior to determining the number of HFU. A 60-min adsorption period was used in both assay procedures. The hemadsorption method allows better definition of an infectious center or plaque than do conventional staining procedures. All virus titers are expressed in terms of HFU. Experimental procedure. Two methods were employed to study infection of cell monolayers by NDV. (i) In the first approach the appearance of hemadsorption foci after various intervals of contact between virus and cells was studied. Virus (0.2 ml) was added to cell monolayers in 60-mm plastic dishes and allowed to adsorb for a suitable period of time. The interaction was stopped by the addition of 2.0 ml of MEM to each dish. This medium was quickly removed and replaced with 5.0 ml of fresh MEM (for HeLa cells) or 5.0 ml of agar overlay mixture (for CEF). The cultures were incubated at 37 C, and the number of HFU was counted at 24 h (HeLa cells) or 48 h (CEF). (ii) The disappearance of virus from the inoculum was used as a measure of virus attachment to cell monolayers. Virus (0.2 ml) was added to each culture. At various intervals, 0.1 ml of the supernatant fluid was removed from the dish and then diluted appropriately. Unattached virus in the supernatant fluid was assayed on a second set of cultures as described above. RESULTS Adsorption of NDV in various media. Experiments were performed to compare the adsorption of NDV to HeLa cells in several media. Dialyzed virus suspensions were diluted in MEM, 0.25 M sucrose, and 0.25 M sucrose containing NaCl or CaCl2. The diluted virus was allowed to adsorb to cell monolayers previously washed with the above diluents. After a 60-min adsorption period at 37 C the unadsorbed virus was removed and fresh MEM was added to all dishes. The results are shown in Table 1. It is evident that efficient adsorption of NDV did not occur in sucrose. Addition of CaCl2 and NaCl greatly facilitated attachment, confirming the observation that ions are necessary for the initial interaction of NDV with cells (12). In our experiments, MEM was found to be a better adsorption medium than the other diluents. Both NDV-C and NDV-H behaved similarly with respect to adsorption in the various diluents. The MEM was also found to be superior to PBS as an adsorption medium (unpublished observations). Effect of adsorption temperature on NDV infection of HeLa cells. Infection of either HeLa cell or CEF monolayers with NDV results in the formation of discrete foci of hemadsorbing cells. The number of such foci in HeLa cells as a function of adsorption temperature at various time periods is presented in Fig. 1. All data were normalized to the 60-min point, i.e., the 60-min value was taken as 100%. The absolute amounts of virus added to cell monolayers were similar for all experiments and usually fell within the range of 100 to 200 HFU per dish. The ability of NDV-C and NDV-H to initiate focus formation on HeLa cells proceeded at the same rate at 37 C. At lower temperatures NDV-H infected HeLa cells more readily than did NDV-C. The relationship between adsorption temperature and HFU is illustrated in Fig. 2 in which 60-min values from several experiments are plotted. It is evident that NDV-C initiates infection of HeLa cells poorly at the lower adsorption temperatures. The relationship between the appearance of hemadsorption foci in HeLa cells and the disappearance of virus from the supernatant at 37 C is presented in Fig. 3. As expected, the amount of unattached virus decreased with time, and the number of HFU increased in a nearly reciprocal fashion. Further, NDV-C and NDV-H did not differ at this temperature. Since the number of hemadsorption foci formed by NDV-C at an adsorption temperature of 22 C was much less than for NDV-H, we examined the rate of removal of virus from the supernatant at this temperature (Fig. 4, left). It is clear that the rate of virus attachment to HeLa cells at 22 C is the same for NDV-C and NDV-H. It is also noteworthy that the rate of attachment of NDV-C and NDV-H at 22 or 37 C is not significantly different (Fig. 4, right). It would appear, therefore, that NDV-C and NDV-H attach to HeLa cells at the same rate but the ability of NDV-C to complete the infectious cycle is impaired. The medium in which NDV was frozen had no effect on the behavior of the virus. When NDV-C was frozen in HeLa cell extracts the number of HFU was not increased on HeLa cell monolayers at a 22 C adsorption temperature (Table 2). Similarly, NDV-H frozen in allantoic fluid did not display a decrease in HFU under the same adsorption conditions. If the suspending media were influencing the differences in HFU observed at 22 C, this experiment should have revealed this. Further support that the initial suspending medium is not involved is presented later. Effect of adsorption temperature on NDV infection of CEF. The data presented thus far suggest that NDV-H infects HeLa cells more a NDV-H was mixed with an equal volume of allantoic fluid and frozen at -20 C for 24 h before dilution and plating on cell monolayers. The same procedure was employed for NDV-C except that HeLa cell extract prepared by freeze-thaw (2 x) of culture was substituted for allantoic fluid. b Adsorption at 22 C, average of triplicate dishes. readily at 22 C than does NDV propagated in chick cells. Reciprocal experiments were performed on chick cells to test whether NDV-C behaves similarly in its homologous host cell. The relationship between adsorption temperature and HFU on CEF is plotted in Fig. 5. Again, 60-min values are plotted with the 37 C values taken as 100%. The results indicate a relationship identical to that shown in Fig. 2 in that the homologous virus infects its host cell more readily than the heterologous virus. In this case, NDV-C was able to initiate focus formation more readily than NDV-H. In an attempt to eliminate the possibility that components in the medium were responsible for the effects described, the same experiment was performed with purified NDV-C and NDV-H. The results shown in Fig. 6 are identical with those for unpurified stock virus. DISCUSSION The entry of enveloped viruses into susceptible cells is thought to occur either by an engulfment process ("viropexis") in which the virus is enclosed in a portion of the cell membrane and is subsequently transported to the interior of the cell (4,19,20), or by a process of membrane interaction in which the envelope of the virus fuses into the cellular membrane with resulting liberation of the nucleocapsid (6,7,15,16). It is probable that both processes are operative. Homologous or heterologous cell components in the viral envelope could influence these entry mechanisms. The data suggest that the cell on which NDV is grown can influence the early stages of virus infection. NDV suspensions from different hosts appear to infect cells with the same facility at 37 C. When virus attaches at lower temperatures, however, subsequent events leading to fruitful infection do not occur as readily with virus propagated on a heterologous host cell. The results are apparently not due to differences in attachment of virus to cells since the amount of NDV removed from the supernatant was the same for both viruses at 37 C or 22 C. It is known that attachment of NDV to cells is not temperature dependent (12). It is unlikely that events after entry are involved since all cultures were incubated at 37 C after the adsorption period. If the uptake of NDV by cells occurs only by phagocytosis, the kinetics of entry of NDV-C and NDV-H should be similar at a given temperature. Less uptake of both viruses would be expected at 22 C, a temperature at which phagocytosis is decreased. The inefficiency of "heterologous" virus entry at the lower adsorption temperature (22 C) suggests that phagocytosis may not be the only process operative. It is possible that the interaction of NDV with its homologous host cell may be more favorable for fusion of the viral envelope with the cell membrane because of similarities in lipid composition (9,23). When homologous NDV interacts with its host cell, the complementary nature of the lipids may facilitate integration of the envelope with the cellular membrane. On a practical level, the results indicate that caution must be exercised in choosing adsorption conditions with paramyxoviruses propagated on different hosts. Whether other enveloped viruses, e.g., herpesviruses, display the same behavior is not known. The data also suggest that enveloped viruses such as NDV may be useful in studies on homologous and heterologous membrane-membrane interactions.

v3-fos

2018-04-03T00:00:38.419Z

1974-03-01T00:00:00.000Z

9096762

s2

Comparison of macroscopic examination, routine gram stains, and routine subcultures in the initial detection of positive blood cultures. Blood was cultured in two vaccum bottles containing Columbia broth with sodium polyanethol sulfonate and CO(2). Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in CO(2)) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture. Blood was cultured in two vacuum bottles containing Columbia broth with sodium polyanethol sulfonate and CO2. Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in C02) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture. There are many methods recommended for the routine-culture and examination of blood samples. There is agreement that blood cultures should be observed at least daily for macroscopic growth, but suggestions as to the need for routine Gram stains and blind subcultures vary from author to author. We are not aware of any published report comparing the efficacy of these procedures in the initial detection of positive blood cultures. Therefore, a comparative study was carried out to assess the value of the three approaches to detection of initial microbial growth in blood cultures. MATERIALS AND METHODS Blood cultures were obtained from patients in the University of Minnesota hospitals (approximately 800 beds) and were processed in the Diagnostic Microbiology Laboratory, which receives about 700 blood cultures per month. Blood was cultured in two vacuum bottles containing 100 ml of Columbia broth with 0.03% sodium polyanethol sulfonate and 10% CO2 (B-D Division of BioQuest). The blood was drawn by physicians, and the amount inoculated into each bottle varied from a few drops to approximately 10 ml. When the bottles were received in the laboratory, filtered air was admitted to one bottle by using a blood collection set (B-D Division of BioQuest); the collection set was removed from the bottle before incubation. The other bottle was considered to be anaerobic. Penicillinase (Difco) was added when indicated. The blood cultures were incubated at 35 C for 7 days or until growth was noted. Cultures from patients with suspected bacte-rial endocarditis or brucellosis were held for 2 to 3 weeks. Cultures were examined macroscopically for growth in the morning and afternoon on the 1st day of incubation and in the morning of each day thereafter. Cultures that appeared positive were Gram stained immediately, and subcultures were made according to the types of organisms seen. Gram stains were performed on all bottles that appeared macroscopically negative on the 1st, 4th, and 7th day of incubation. Blind subcultures were also made on the 1st and 4th days to a sheep blood agar plate (incubated anaerobically) and to a chocolate agar plate (incubated in C02). Subculture plates were held for 2 days before they were discarded as negative. Each procedure was performed in the routine laboratory by a total of 13 microbiology technologists on a rotation basis. RESULTS The method of first detection of growth is shown in Table 1. There were a total of 7,357 blood cultures examined over a period of 10.5 months, and 1,127 were positive. Of these, 734 Table 2 shows the day on which cultures were noted to be positive by three methods of detection. Forty-seven percent of those first detected by macroscopic examination were found on the 1st day. Of those first detected by Gram stain, 49% were found on the 1st day, 28% were found on the 4th day, and 23% were found on the 7th day. Of the positive cultures first detected by subculture, 76% were detected on the 1st day and 24% were detected on the 4th day. One hundred twenty-five positive cultures were not apparent macroscopically on the 1st day, and 106 positive cultures were not detected by Gram stain on the 1st day, nor were 33 positives detected by Gram stain on the 4th day. Of the 1,127 positive blood cultures, 467 (41.4%) were detected on the 1st day either macroscopically or by Gram stain. The numbers and types of organisms isolated, along with the mean times for detection (by all methods) are shown in Table 3. Of all the organisms isolated, Haemophilus influenzae, H. parainfluenzae, Moraxella sp., and Neisseria gonorrhoeae were detected first only by subculture. These organisms were never detected first by macroscopic examination or Gram stain, although approximately one-half of the Haemophilus cultures appeared macroscopically positive subsequent to subculture. Of the Pseudomonas aeruginosa isolated, only one-third were detected first macroscopically, one-third were detected first by Gram stain, and the remaining one-third were detected first only by subculture. Anaerobic organisms were almost always de- tected either by macroscopic examination or Gram stain. Only four strains of Bacteroides were first detected on the anaerobic subculture plate. The organisms detected first by the 7th-day Gram stain included Propionibacterium acnes, Candida, Corynebacterium, Peptococcus, Pseudomonas, Staphylococcus epidermidis, and Torulopsis glabrata, although some strains of these bacteria were also detected by the other methods. DISCUSSION The data presented indicate that, for optimal speed in detection and identification of organisms from positive blood cultures, both routine Gram stains and blind subcultures should be performed in addition to daily visual inspection of cultures. If routine Gram stains and subcul-INITIAL DETECTION OF POSITIVE BLOOD CULTURES addition to macroscopic inspection, only Gram stains were done, there would have been a delay in 12% of the cultures. If only subcultures had been performed, 23% of the positive reports would have been delayed at least 1 day. One might make the point that results of subcultures themselves were delayed by 1 day and that the culture in some cases may have been positive macroscopically the next day; however, even though this may be true, at the time of reading the subcultures a more definite identification of the organism could be given to the physician rather than just its Gram stain morphology. Subcultures were especially important in the more rapid detection of Haemophilus, since these organisms were all detected first only by this means. Both Gramstains and subcultures were also valuable in the more rapid detection of Pseudomonas, as two-thirds of those isolated were detected first only by Gram stain or subculture. Our experience with Pseudomonas bears out the study by Slotnick and Sacks (3), who stated that the use of visible growth or Gram stains alone are not sufficient to detect the presence of Pseudomonas in blood culture media. Although there is no question about the importance of a Gram stain to detect positive blood cultures on the 1st day, the value of Gram stains on the 4th day in relation to the amount of work involved and the clinical importance might be questioned. In this study, approximately 6% of the positives were first detected by Gram stains on the 4th day. Individual judg-ments would have to be made as to whether detection of the positive on the 4th day would be that much more important than detection by subculture the following day. The blood cultures in this study were incubated for a maximum of 7 days, except in cases of suspected brucellosis or endocarditis. This incubation period was based on the results of previous unpublished studies in our laboratory which demonstrated the rarity of isolation of clinically significant organisms after 1 week of incubation. Effersoe (1) has also shown that incubation for longer than 7 days is not necessary, especially if "control" Gram stain and subcultures are performed. It was not the intent of this study to assess the overall rapidity of organism detection. However, the information in Table 3 does allow for comparison with other recently published studies (2, 4) on this subject. On the basis of these comparisons, we feel that the spacing of the procedures evaluated in our study are appropriate and practical for the clinical laboratory.

v3-fos

2020-12-10T09:04:22.666Z

1974-08-01T00:00:00.000Z

237230515

s2

Quantitative Infrared Photoanalysis of Selected Bacteria A technique to measure transmitted infrared radiation from minute biological systems is described. Infrared color film was exposed by radiation transmitted through bacterial colonies. The resultant photographic image was unique for each species of bacteria examined and spectral analysis of the image provided differential light emission patterns which could be quantitated. A formula for developing numerical comparisons among bacterial colonies was provided. The results of this numerical procedure gave quantitative relationships for the total infrared data from each microbial colony and made possible the differential identification of ten species of medically significant bacteria. A technique to measure transmitted infrared radiation from minute biological systems is described. Infrared color film was exposed by radiation transmitted through bacterial colonies. The resultant photographic image was unique for each species of bacteria examined and spectral analysis of the image provided differential light emission patterns which could be quantitated. A formula for developing numerical comparisons among bacterial colonies was provided. The results of this numerical procedure gave quantitative relationships for the total infrared data from each microbial colony and made possible the differential identification of ten species of medically significant bacteria. Of all differences among bacterial species, none are more generally relied on for identification than cellular morphology and metabolism (2). These characteristics are demonstrated through variations of colony color, texture, density, geometry, rate of growth, gas evolution, heat liberation, and chemical composition. The collective effect of these features produces unique patterns of absorption or reflectance in response to interaction with the electromagnetic spectrum (3,4). A number of studies (8,9) have shown that infrared spectrophotometry can be used to distinguish between various genera of bacteria. However, these procedures are time consuming and demand specialized equipment not readily available in nonresearch and clinical laboratories. This paper describes results of an infrared analysis of bacterial colonies which may have future-application in the clinical laboratory. MATERIALS AND METHODS Organisms and media. Bacteria from the Brigham Young University culture collection were maintained on agar slants consisting of 12% beef heart infusion (Difco), 1.2% peptone, 1.2% gelatin, 0.6% dextrose, 0.6% casein, 0.5% disodium phosphate, 0.4% sodium citrate, and 0.9% agar. The transparent medium used to obtain transmitted infrared (TIR) data consisted of 1.6% nutrient agar, 0.25% yeast extract (Difco), and 0.25% glucose. A 20-ml amount of medium was placed into disposable petri dishes (100 by 15 mm). The medium was inoculated with approximately 50 bacterial cells of the desired strain, and the cells were distributed evenly over the agar surface. All cultures were incubated at 37 C. Infrared photomicrography. Figure 1 is a sche-matic diagram of the instrumentation used for infrared recording of bacterial colonies. A Leitz Photolab II photomicroscope was fitted with a 35-mm camera back and further modified as follows. (i) Light eminating from the tungsten source was enclosed in a light-tight corridor to the microscope stage ( Fig. 1, item E). (ii) The stage was modified such that light filters could be imposed between the light and the object (Fig. 1, item D). A Leitz brightfield microscope condenser was used with both aperture diaphrams wide open. One eyepiece was fitted with a micrometer for centering the object and measuring bacterial colonies under examination. A Kodak (Rochester) number 87 infrared filter was positioned in the light path between the source and the object (Fig. 1, item D). This filter allows transmission of radiation from 720 nm to beyond 1,500 nm. Power to the light source was controlled by using a filament transformer equipped with an ampmeter and voltmeter. Ektachrome infrared film (Kodak) having a single emulsion series number was stored at 4 C in moisture-tight containers prior to use. Infrared recording procedures. IR film to be standardized was allowed to come to room temperature (22 C) before placing it in the camera back. The power source was equilibrated and a petri dish containing culture media was placed on the microscope stage. The microscope was focused on the agar surface through the camera window. The power was adjusted to 1 A and the film was exposed by using an A.S.A. of 100 and a shutter speed of 1/8 s. Subsequent exposures were made at 0.25 A power increments up to 8 A. The film was then developed and used to demonstrate film characteristics (indicated in Fig. 3, 4, and 5). Each subsequent role of film was compared to this standard to insure proper control of exposure and development procedures. Each roll of film must be standardized to correct for variation in the media and for photographic procedures. This standardization is accomplished by re-205 moving the number 87 filter and focusing the optics on the agar surface. The filter is replaced and two frames of film are exposed, one at 3.5 W of power and the other at 8 W. When these control frames have been exposed the filter is again removed and the bacterial colony to be analyzed is brought into focus under the microscope. The filter is replaced and final focusing is done through the camera by using the colony edge for proper planar adjustment. Because colony morphological detail is not required, it is unnecessary to make focal length adjustment for changes in colony height. The power is adjusted to a value between 6.5 and 8.0 W to obtain proper exposure of the film, and the colony is photographed. Development of the film is done immediately after exposure. Exposed IR film was developed by using Kodak E-4 processing (6). Fresh Kodak developer solutions were used to develop only four rolls of film and then were discarded. Constant film-chemical agitation was maintained during the development procedures. Recommended film development times and water bath temperatures were held constant (7). A 10-mm wide strip was cut from the center of the photograph of the desired colony. This picture was then attached to a 35-mm photographic film leader 10 cm long and 10 mm wide. The sample and leader were then threaded into the scanning assembly of a Beckman DBG spectrophotometer (Fig. 2). The spectrophotometer was fitted with a 25-cm chart recorder set to linear response. With the light wavelength set at 580 nm and by using a beam width of 1.5 mm, the sample was passed in front of the light beam at 2.5 cm/min. The density-geometry pattern of the photographed colony was recorded on the chart paper. For infrared analysis, the scanner as shown in Fig with the light wavelength set at 760 nm, the wavelength scan is activated and completed at a wavelength of 380 nm. This procedure is, in principle, a comparison between two infrared (IR) photographs of identical agar surfaces, one of which includes a bacterial colony. The difference in appearance, as seen be the IR film, provides a means for characterization of the colony. This difference is measured by using a scanning spectrophotometer to reveal subtle changes in the layers of dye on the film. RESULTS Kodak Ektachrome IR film contains three layers of visible and infrared-sensitive film dyes. Exposure results in red coloration as a function of the incident infrared intensity. IIEJ4j5I 1 206 Therefore, this film provides both color and 0 density change as a function of the infrared properties of the photographed bacterial colonies. Figure 3 shows analysis of a plain transparent agar plate photographed at two infrared intensities. The data from such samples are in TO response to the transmitted IR which has only been attenuated by the transparent agar. These data serve as standards for peak positions and 60 intensities which can then be used for analytical 2 comparisons. Two characteristics result from analysis of these recordings: (i) peak area, which is designated as the static infrared unit (SIRU) and is determined by dividing the area 40 under the peak (Fig. 3, peaks 1 gamma value is characteristic for individual emulsions and experimental conditions and should be obtained for each roll of film that is used. Should a recording be made at a variety of infrared intensities on a particular roll of film, any standard SIRU value can be found from this data. As each roll is its own reference, the significance of the control standard is to give an indication of how far from an optimal reproduction any particular film roll is. Data obtained by this technique of transmitted IR recording provides two types of information about the bacterial colonies. (i) a spectrophotometric scan of the photographed bacterial colony gives information which is highly characteristic for many species. (ii) Analysis of the photographs of bacterial colonies gives an indication of the alterations in the IR radiation that is absorbed by the colonies as compared to the unattenuated IR radiation of an appropriate agar standard. Fig. 6 and 7A) result from differences in colony geometric configuration and are obtained by passing the film through the spectrophotometer operating at the indicated wavelength. The transmission recordings ( Fig. 6 and 7B) are obtained by keeping the film stationary in the spectrophotometer and scanning the photograph over a range of wavelengths of light. In these figures it should be kept in mind that the peak areas represent the greatest cell density in the colonies. The recorded IR pattern in these figures is mediated by colony composition as it interacts with radiation from the illuminating source. Figure 8 shows the changes in infrared transmissions of bacterial colonies as a function of incubation time. The change in infrared transmission with age indicates change in geometry, and composition which collectively affect the colony's ability to absorb, transmit, and reflect IR radiation. However, such transmission change was not obvious for all of the organisms tested. Much of the final characteristic of the transmission patterns was not developed until late in the colony growth process. It was also I .;e. -k-208 APPL. MICROBIOL. I I10 apparent that reproducibility and ease of species identification increased with the age of the colony. The transmission and geometric patterns provide three characteristics which are used to determine quantitative values: (i) geometric conformation ( Fig. 6 and 7A); (ii) the SIRU Fig. 6 and 7B); and (iii) peak positions ( Fig. 6 and 7B). Using the values from these three characteristics it is possible to construct a table showing quantitative relationships for each species of organism examined (Table 1). In Table 1, two characteristics of the infrared transmission recording are combined into one value called beta. The position of peaks 1 and 2 as well as the SIRU for each peak is determined from the recorded transmission curve (Fig. 6 and 7B). The values thus obtained are used in the following formula to obtain beta. , = (xs) /(ty) where ft equals beta value; x equals (SIRU of peak 2 from the standard) + (wavelength of maximum transmission of peak 2 from the standard); s equals (SIRU of peak 2 from the test organism) + (wavelength of maximum transmission of peak 2 from the test organism); t equals (SIRU of peak 1 from the test organism) + (wavelength of maximum transmission of peak 1 from the test organism); and y equals (SIRU of peak 1 from the standard) + (wavelength of maximum transmission of peak 1 from the standard). The structure of the equation was derived in its present form as a result of attempts to determine the ratio most useful in indicating maximum differences between sample values and suggests no other relationships. The base of the peak in millimeters produced by a 580-nm scan of a microbial colony photograph (see Fig. 6A). b Alpha: First number represents the number of transmission peaks obtained on photoanalysis (refer to Fig. 6A), the slant has no mathematical meaning, whereas the second number equals the base times the area of the peak divided by 100. c Peaks 1 and 2 refer to the transmission recording peaks (see Fig. 6 and 7B). d SIRU: static infrared unit; A: wavelength of peak transmission. "Beta: a value derived according to the formula on page 209. ' SD, standard deviation of beta from five experimental measurements. DISCUSSION The data presented in this study have demonstrated that bacterial colonies, even of closely related species, possess sufficient structural and compositional differences to provide characteristic IR interactions. These characteristics can be recorded on IR-sensitive film. Subsequent spectrophotometric analysis of the film provides data useful in quantitating specific differences. The colony's infrared transmission response suggests a dependence on the collective effect of colony density, color, geometry, and chemical composition. IR transmitted through the bacterial colonies has the capacity to reduce dyes in the film which are IR wavelength specific. The reduction of these selective dyes is characteristic of the colony through which the IR radiation has passed. The data presented in this paper are easily obtained, require limited specialized equipment, and may be feasible in a clinical as well as research setting. Control experiments using white light and noninfrared Ektachrome film have failed to produce similar geometric data or light interaction data which could allow bacterial identification (Fig. 9). The overall effect of the inherent bacterial IR emissions (passive infrared) incident on the film is difficult to appraise. E. coli produces about 1.4 x 10-5 W of IR per s per colony area when ideal emissivity conditions exist (1, 3), whereas Staphylococcus aureus produces only about 9.0 x 10-6 W of IR per colony area per s. However, currently available IR film requires a minimum of about 3.0 x 10 2 W of IR per colony area per s, in order for the film to be exposed sufficiently to be detected by photoanalysis (4,5). Therefore, the inherent IR of the colonies is not sufficient to effect a response on the film due to their low power and the long wavelength of such passive emissions. These limitations in detection of passive bacterial IR require that a carrier infrared source be supplied, such as the photomicrographic system used in these studies. Due to the low sensitivity of the film and short IR wavelength required for exposure, it is doubtful that the small but characteristic amount of passive infrared emission from the bacterial colonies contributes significantly to the overall transmission response of the test organisms. The data contained in Table 1 do not suggest that any interpolations can be made regarding specific information on chemical composition of the cells. Variations in the system such as film emulsion changes, changes in film developing chemicals, compositional changes in bacterial growth media, and different microphotographic systems prevent the data in Table 1 from being established as standard values for the organisms shown. With future refinement and standardization of media and film development, it may well be possible to establish a reference by which unknown organisms could be identified by using this infrared transmission procedure.

v3-fos

2018-04-03T03:32:10.390Z

1974-05-01T00:00:00.000Z

6519445

s2

Microculture system for detection of Newcastle disease virus antibodies. A microculture system utilizing cytopathic effect (CPE) and hemadsorption (HAd) end points was effective in determining the level of Newcastle disease virus (NDV) antibodies. The microculture system was of comparable sensitivity to the plaque reduction test for the detection of NDV antibodies. The standards by which the CPE and HAd microculture tests would be considered reproducible were defined. The results indicate that the CPE and HAd microculture tests are reproducible within one twofold dilution. In recent years, microculture methods for virus titration and serological procedures have come into more frequent use. The application of microculture has been reported in the study of arboviruses (2), transmissible gastroenteritis virus (11), rubeola virus (6), poliovirus (5), respiratory virus seroepidemiology (8), and in other serological investigations (3,4,9). Methods utilizing microculture have been shown to be as sensitive and far more economical than macro cell culture methods (4). Laboratory procedures used to detect Newcastle disease virus (NDV) antibodies include the hemagglutination-inhibition (HI) test, neutralization in embryonated eggs, plaque reduction, and the egg-bit technique (1). The detection of HI antibodies and virus neutralizing (VN) antibodies has been utilized as an indication of exposure to NDV. Serum neutralization tests are universally accepted as standard quantitative tests for antibody levels. However, the neutralization tests for NDV, by using either embryonated eggs or cell cultures as a virus indication system, are expensive and cumbersome. The objective of this study was to develop a virus neutralization test for NDV antibodies utilizing microcultures as the indicator system for unneutralized virus. MATERIALS AND METHODS Media. Growth medium for cell cultures consisted of Hanks balanced salt solution supplemented with 0.25% lactalbumin hydrolysate, 10% fetal calf serum, and 10% tryptose phosphate broth. Penicillin and streptomycin were added at concentrations of 100 U/ml and 100 Ag/ml, respectively. Virus dilutions were prepared in Hanks balanced salt solution. Agar overlay medium, for plaque enumeration, consisted of Eagle minimal essential medium supplemented with 2% fetal calf serum and 1% purified agar (Difco Laboratories). Cell cultures. Chicken kidney cell cultures were used for virus propagation. Kidneys from 1-day-old chicks were trypsinized for 30 min, and the dispersed cells were filtered through sterile gauze and sedimented by centrifrigation. A 1-ml amount of packed cells was suspended in 200 ml of growth medium. Source of virus. The Kansas-Manhattan strain of NDV was supplied by P. D. Lukert, University of Georgia. This strain was selected because of its rapid cytopathic effect (CPE) in chicken kidney cell cultures. Serums. Serums used in the experiments were obtained from 34 individual chickens. Thirty serum samples were obtained from broiler chickens in northern Georgia. The broilers were given NDV, B-1 vaccine at 7 days of age by aerosol; serum samples were collected 42 days later. Four negative serum samples were obtained from unvaccinated specific pathogenfree chickens housed at the Poultry Disease Research Center, University of Georgia. To avoid bias, all serum samples were randomly coded before each test. In this manner, the sera were examined in a sequence unknown to the individual conducting the tests. The titration end points were not decoded until the data were ordered for statistical analysis. Virus-neutralization tests: plaque reduction. Neutralizing activity of sera by plaque reduction was determined by the constant virus serum dilution technique. A virus dilution shown to give approximately 100 plaques was mixed with an equal volume of each serum dilution. Plaques were counted after incubation for 48 h, and the highest serum dilution that gave greater than 50% plaque reduction as compared with virus control plates was recorded as the end point. Microculture cytopathic effects. Microculture plates (IS-FB-96-TC, 0.4 ml/well, Linbro Chemical NDV ANTIBODY DETECTION SYSTEM Co., New Haven, Conn.) were used for determination of virus neutralization by inhibition of viral CPE. Twofold serum dilutions from 1: 10 to 1: 1,280 were prepared in tubes. The serum dilutions were added to equal volumes of NDV so that each 0.05 ml of virus-serum mixture contained 1,000 to 2,000 plaqueforming units (PFU) of NDV. The virus-serum mixtures were mixed in a test tube and allowed to react for 30 min at 26 C. Microculture plates were inoculated with 0.05 ml of the virus-serum mixtures. Each virus-serum dilution was inoculated into eight replicate well cultures. Five uninoculated replicate well cultures served as cell controls. Virus controls consisted of inoculating five replicate well cultures (Fig. 1). After the inoculation of the virus and virus-serum mixtures, each well was inoculated with 0.15 ml of the chicken kidney cell suspension. The microculture plates were then sealed with a clear mylar sheet with an adhesive back (35 PSM, Linbro Chemical Co., New Haven, Conn.) and covered with a clear polystyrene top (53, Linbro Chemical Co., New Haven, Conn.). The microculture plates were incubated at 37 C for 48 h. The incubation period, temperature of incubation, and PFU of NDV utilized in the test were determined by preliminary trials to obtain maximal sensitivity in the shortest period of time. After incubation, the medium was removed, and the cells were fixed with 10% neutral Formalin for 3 to 5 min. The Formalin was removed, and the fixed cells were stained with 1% crystal violet for 30 min. The stained cells were examined by gross inspection with an appropriate light background. Control monolayers and virus negative monolayers appeared solid blue. Virus-infected monolayers were mottled or had discrete plaques (Fig. 1). The antibody titers obtained in the microculture CPE system were calculated by the Kiirber method (7). Microculture hemadsorption. Hemadsorption (HAd) tests were conducted in the same type of plates used for the microculture CPE test (Fig. 1). In this case, 0.05 ml of chicken erythrocytes were added to three replicate cultures which had been inoculated with virus-serum mixture, to five replicate cultures used for virus controls, and to five replicate cultures used for cell controls. The red blood cells were allowed to settle at 4 C. The wells were washed two times with phosphate-buffered saline and examined microscopically for HAd. The antibody end points were determined as the reciprocal of that serum dilution which completely inhibited the adsorption of chicken red blood cells. Reproducibility of antibody titers in microculture systems. The reproducibility of antibody titers obtained in the microculture systems was determined by repeating the microculture CPE and HAd tests with 25 of the serum samples three times at intervals of 4 months and 1 week. Statistical analysis. Assessment of the accuracy of the CPE and HAd microculture tests requires a knowledge of the true neutralizing antibody titers in CPE and HAd microculture systems. This information is not available. Therefore, the question of accuracy cannot be answered directly. However, if results of a test system are consistently reproducible, accuracy can be inferred. Because the true CPE and HAd microculture titers remain the same, only the variations (human and mechanical) inherent in any test system need to be considered to answer questions concerning reproducibility. The mean and the standard deviation are the "best" estimates of variation (10). By utilizing these statistics a "reproducibility" level can be defined. An acceptable level of reproducibility of many serological tests is, by custom, commonly referred to as being "within" one twofold dilution. By employing the statistics which can be developed and the presently accepted custom, two hypotheses are advanced. (i) The CPE and HAd microculture tests are reproducible between one twofold dilution (Fig. 2). (ii) The CPE and HAd microculture tests are reproducible within one twofold dilution (Fig. 2). The criterion of reproducibility revolves around "between" and "within" one twofold dilution. The logarithmic range between one twofold dilution is 0.602 and within one twofold dilution is 1.204 (Fig. 2) on May 8, 2020 by guest http://aem.asm.org/ Downloaded from confidence interval of the means of the CPE and HAd microculture titers do not exceed 0.602 (between) or 1.204 (within). From the logarithmic numbers obtained from the end point determination, the mean, standard error, coefficient of variation, and confidence interval for the CPE and HAd microculture titers of each serum sample were calculated (10). The confidence interval based upon the t-distribution was calculated at the 90 and 95% levels. RESULTS The neutralizing antibody end points in microculture CPE and HAd systems were comparable to those obtained by the plaque reduction technique. The results are shown in Table 1. The reproducibility of the microculture CPE and HAd tests was established by utilizing the t-distribution. Confidence limits for serum sam-APPL. MICROBIOL. ple antibody titer means were calculated at the 90 and 95% levels. At the 90% confidence interval, it was ascertained that the range for the CPE microculture test did not exceed the standard of reproducibility of being between one twofold dilution in 17 of the 25 sera tested. Additionally, all of these confidence intervals were within one twofold dilution (Table 2). It was also determined that at the 95% confidence interval this range for the CPE microculture test exceeded the standard of reproducibility of being between one twofold dilution in 10 of the 25 sera. However, when the standard of reproducibility was to be within one twofold dilution, only 3 of the 25 sera failed to meet this standard. The coefficient of variability is another method of describing variation in a population. 80 80 20 40 320 20 0 80 160 -80 0 320 160 40 80 80 80 80 160 0 160 40 320 160 80 80 20 0 320 80 640 80 320 80 80 160 20 20 320 20 0 160 160 160 0 320 160 40 80 160 20 160 320 0 320 80 640 160 160 160 20 0 640 In this case, in the three CPE microculture titers of each serum sample, the average coefficient of variability was 6%. It should be noted that sera 6, 24, and 27 had coefficients of variability of 12.7, 9.9, and 16.1%, respectively. These were the three largest coefficients of variability, and in each case the confidence interval (95%) exceeded the standard set forth for reproducibility ( Table 3). Nine of the sera examined in the HAd system had the same titer in each of the three trials. Each of the remaining 16 sera examined had two identical titers, and the third differed by only one twofold dilution (Table 1). Of the sera which had the same titers for each trial, the standard error of the mean titration was, of course, zero and all met the reproducibility levels at any of the standards previously described. For the other 16 sera the calculated logarithmic standard error of the mean was 0.1003. Therefore, the confidence interval at the 90% level was 0.2935 and the confidence range was 0.5870 (see legend, Table 2). At the 90% confidence level these sera did not exceed the reproducibility standard either between or within one twofold dilution. Similarly, the confidence interval at the 95% level was 0.4316, and the confidence range was 0.863 (see legends, Tables 2 and 3). At the 95% level, all of these sera failed to meet the reproducibility level of being between one twofold dilution, but all met the standard of being within one twofold dilution. The average coefficient of variation of the 25 titers in the HAd system was 4.8%. DISCUSSION The results presented indicate that the microculture CPE and HAd test systems were of comparable sensitivity to the 50% plaque reduction test for detecting neutralizing antibodies to Antibody end points in the HAd system were determined as that serum dilution which completely inhibited the adsorption of chicken red blood cells. The small variation in the sensitivities of the microculture CPE and HAd tests and the plaque reduction test may be due to the method of reading the end points. The specificity of the microculture system was tested by the blind inclusion of four sera derived from specific pathogen-free chickens, in which cases all tests were negative. The microculture method, in addition to being very sensitive in detecting NDV antibodies, has the advantage of being economical, because the quantity of kidney cells harvested from a single 1-day-old chicken is sufficient to establish microcultures in approximately 300 microculture wells. Because CPE are not measured by microscopy examination, but by gross visual inspection of plates, the microtiter CPE method requires less time, and there is less chance for subjective error in reading CPE end points. It was stated earlier that knowledge of the true CPE and HAd microculture titers of any serum is not available. The probability statements state that we are either 90 or 95% confident that we have included the true mean within the confidence limits outlined. For the CPE microculture test to be considered reproducible, the standard is that the calculated confidence interval range must either be between or within one twofold dilution. At the 95% confidence interval range, 22 of the 25 confidence interval ranges fell within one twofold dilution. This assessment of reproducibility seems rigorous since only three replicates were employed in arriving at a sample mean and standard deviation. Significant variation in any one of the calculated CPE microculture titers was enough to extend the confidence interval beyond the criteria stated to be acceptable as being reproducible. It would appear then that the human and mechanical variation inherent in any serological test is minimal in the CPE microculture test. This is further borne out by the calculated coefficients of variation which describe the amount of variation in a sampled population. The average titer variation of the sera sampled utilizing the CPE microculture test was 6%. Because individual investigators have varying requirements for reproducibility levels, no criteria for accepting or rejecting the two hypotheses stated earlier were delineated. However, in the case of the CPE microculture system, it would seem that the first hypothesis concerning reproducibility between one twofold dilution should be rejected. In this case, the calculated confidence interval range at 90 and 95% exceeded the reproducibility standard in 8 and 15 sera, respectively. The second hypothesis concerns reproducibility within one twofold dilution. At the 90% level, the confidence interval met the reproducibility standard in all cases. At the 95% level, the confidence interval met the reproducibility standard 22 out of 25 times. The chi-square criterion (corrected for continuity) was applied to determine if this result could have happened by chance alone. The calculated P of >0.005 made this seem unlikely. It would appear then that the second hypothesis can be accepted conditionally upon the standards of reproducibility required. For the HAd test, assessment of reproducibility was again rigorous because of the minimal number of replications employed. This situation is further complicated because the accepted method of measuring titers is crudely quantitative. Therefore, in a set of three titration end points, if two of the end points are the same and the third is different only by one twofold dilution, only the 95% confidence limits of being reproducible within one twofold dilution are easily met. Slight deviation of end points either sequentially (1: 20, 1:40, 1:80) or differing by a fourfold dilution (1: 20, 1: 20, 1: 80) lowers this estimation of the 90% confidence limits of being reproducible within one twofold dilution. These deviations did not occur, and it would appear that extraneous variation is minimal in the HAd test. This statement is supported by the average coefficient of variation of the titers which is 4.8%. At the 90% confidence level, the HAd test met the reproducibility standard in all cases. Therefore, the first and second hypotheses can be accepted if this level of confidence is acceptable. At the 95% confidence level, only 9 of the 25 sera met the reproducibility standard of being between one twofold dilution, whereas all were reproducible within one twofold dilution. At this level of confidence it would seem that the first hypothesis should be rejected and the second accepted. LITERATURE CITED 1. Beard, C. W. 1969. The egg-bit technique for measuring Newcastle disease virus and its neutralizing antibodies.

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2018-04-03T04:18:46.374Z

1974-01-01T00:00:00.000Z

38552403

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Reaction mechanisms of thiamine with thermostable factors. Distributions of thermostable thiamine-inactivating factors in about 10 kinds of ferns and brackens and 42 kinds of other plants were investigated. It was observed that besides ferns and brackens, some plants (most of them greenish or yellowish in color) had thermostable thiamine-inactivating activities, and that shiitake, okra, fukinoto (butterbur flower stalk), diluted water extract of green tea, and diluted coffee had activities which oxidized thiamine into thiamine disulfide. The present study showed that one of the factors isolated from fern 3, 4-dihydroxycinnamic acid (caffeic acid) and catechol distributed in plants accelerated decomposition of thiamine at pH 7-7.5 into 2-methyl-4-amino-5-aminomethyl-pyrimidine, decomposing the thiazole moiety, probably into γ-aceto-γ-mercapto-propylalcohol and formic acid. However, some flavonoids, especially 6, 7, 4'-trihydroxyisoflavone (Factor 2) were proved to have the ability to accelerate oxidation of thiamine into thiamine disulfide at pH 7.5. Estimation was made of the decomposition products of thiamine with caffeic acid, catechol and Factor 2 under certain conditions. Distributions Since WESWIG et al. (1) reported the existence of thermostable thiamine -inactivating factors in fern, the fact has been confirmed by many workers. From the experimental results of these workers it has been considered that ferns contain not only thermostable factors but also the thermolabile non-dialyzable thiaminase. NAKABAYASHI (2) isolated two flavonoid pigments, astragalin and isoquercitrin, from bracken and proved the presence of rutin as the so-called "thermostable antithiamine factor." HASEGAWA et al. (3) also suggested that the thermostable thiamine-decomposing factors of fern are flavonoids. They studied the activities of various flavonoids as well as phenol derivatives. Flavonoids possessing o-diphenol in the side chain were found to be most active. SAKAMOTO and FUJITA (4) isolated isoquercitrin as a thermostable thiamine-decomposing sub-stance from sweet potato leaves. HOSODA (5) obtained a crystalline product from the reaction mixture of thiamine with catechol or hydroquinone at pH 7, and proved that the crystals were not only identical with the crystals isolated from thiamine reacted with rutin by HASEGAWA et al. (6), but also identical with thiamine disulfide. Later, BERUTTER and SOMOGYI (7) isolated 3, 4-dihydroxycinnamic acid (caffeic acid) from fern as one of the thermostable antithiamine factors in plants. SOMOGYI and BONICKE (8) and DAVIS and SOMOGYI (9) attempted to clarify the reaction mechanism of the inactivation of thiamine by caffeic acid and other orthodiphenols and reported that the course of the reaction was biphasic, an observation which we could not confirm under our experimental conditions. The reaction was made partially reversible by the addition of cysteine (or other reducing substances) to the assay mixture, indicating that some thiamine disulfide was formed. The latter reaction has been observed by DAVIS and SOMOGYI (9) and the present authors. However, the structures of other forms , of thiamine made inactive by the thermostable factor have not yet been disclosed. We have investigated the mechanism of thiamine inactivation by the thermo stable factors; caffeic acid, catechol, and flavonoids present in plants. Experi ments and results are presented here. EXPERIMENTAL 1. Test for the thermostable thiamine-inactivating factor in plants 1) Extraction of factors. Samples of fresh plants or dried powdered ferns were homogenized with 10-20 volumes of distilled water and boiled for 30 min to extract thermostable factors and inactivate thiaminase. The water extract was centrifuged to obtain a clear supernatant.2 ) Reaction of thiamine with the extract. Conditions for the reaction of thiamine with the extract and determination of the remaining thiamine and thiamine disulfide (SSB1) are shown in Table 1. Distribution of the thermostable thiamine-inactivating factors About 50 kinds of plants including ferns were tested to investigate the ac tivity of the thermostable thiamine-inactivating factors. The results are shown in Tables 2 and 3. From these tables it is seen that some of the plants-shiitake, okra, butterbur flower stalk (fukinoto), black tea, and coffee-have factors which primarily produce the oxidized form of thiamine (thiamine disulfide) from thia mine. Extracted factors from ferns showed some ability to produce thiamine disulfide (in amounts from 2 to 27% of the thiochrome negative form of thiamine). Thiamine-decomposing activity of caffeic acid The results of experiment under different conditions are shown in Tables 4 and 5 and Figs. 1, 2, and 3. From the data in the Tables and Fig. 1, it is obvious that production of thiamine disulfide is not consistently large. As shown in Fig. 3, the course of the thiamine decomposition estimated by the thiochrome method is almost identical with that estimated by the diazo method. This means that the decomposition mechanism of thiamine is not deamination of pyrimidine moiety of thiamine by caffeic acid. If the value estimated by the diazo method on the reaction mixture of thiamine and caffeic acid is higher than that by the thiochrome method, it may be possible that deamination has occurred in the pyrimidine moiety of thiamine. 3. Ability of caffeic acid to destroy thiamine-analogues Reactions of OHB1, HET, and HPT with caffeic acid were compared with that of thiamine as described above. As shown in Fig. 4, thiamine is more sensi tive to decomposition with caffeic acid than are the analogues tested. as 5 and 6 in Fig. 8. As seen in the chromatograms of reaction mixtures of thiamine with caffeic acid or catechol, the thiazole moiety of thiamine was not detected, but the pyrimidine moiety was indicated by a spot with an Rf value iden tical with that of 2-methyl-4-amino-5-aminomethyl-pyrimidine (Pm-CH2-NH2), not with 2-methyl-4-amino-5-hydroxymethyl-pyrimidine (PM-CH2OH). In the case of the reaction mixture with Factor 2, however, a major spot of thiamine disulfide was detected, but neither a pyrimidine moiety nor a thiazole , moiety was detectable. This result corresponded with evidence for the formation of thiamine disulfide in the reaction mixture of thiamine and Factor 2 shown in Fig. 7. The parts of the paper strip corresponding to the spot with Rf 0.53 of the reaction mixture of thiamine and caffeic acid or catechol and that of Pm-CH2-NH2 as a standard were extracted with acidified water and the UV spectra measured at pH 2 and pH 9. The extracts from the reaction mixtures showed almost identical curves with the standard at pH 2 and 9, respectively, as shown in Fig. 9. the chromatograms. Extracted samples of paper strips subjected to the reaction mixture were compared with those of standard curves and the total amounts in the reaction mixture calculated. As shown in Table 7, around 10% thiamine di sulfide and 70% Pm-CH2-NH2 and trace amounts of thiochrome were produced from thiamine with caffeic acid or catechol under the above reaction conditions. On the other hand, about 70% was thiamine disulfide in the reaction mixture of thiamine and Factor 2; Pm-CH2-NH2 was difficult to detect from the mixture on the paper partition chromatogram. and caffeic acid or catechol, strongly supports the presumption that the mechanism in this case is the same as that described by MATSUKAWA et al. (11) . On the other hand, there are also many factors in nature, such as flavonoids, which ac celerate mainly or partly the oxidation of thiamine into thiamine disulfide, as indicated in the case with 6, 7, 4'-trihydroxyisoflavone, quercetin and rutin.

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2020-12-10T09:04:20.551Z

1974-11-01T00:00:00.000Z

237234455

s2

Aspergillus flavus Infection and Aflatoxin Production in Corn: Influence of Trace Elements Distribution of trace element levels in corn germ fractions from kernels naturally infected with Aspergillus flavus and from kernels free of the fungus demonstrated an association between the presence of A. flavus and higher levels of metals. A. flavus production of aflatoxin on various autoclaved corn media showed that ground, whole corn was an excellent substrate; similar high levels of toxin were observed on full-fat corn germ but endosperm and defatted corn germ supported reduced yields. The influence of trace elements and their availability in defatted corn germ to A. flavus-mediated aflatoxin biosynthesis were measured. Enrichment of the substrate with 5 to 10 μg of manganese, copper, cadmium, or chromium per g of germ increased toxin yields. Addition of lead or zinc (50 to 250 μg/g) also enhanced toxin accumulation. Aflatoxin elaboration was reduced by the addition of 25 μg of cadmium per g or 500 μg of copper per g of germ. germ. Fennell and co-workers (6) found that Aspergillus flavus was primarily associated with the germ region of infected corn kernels. Since corn germ contains lipids, proteins, carbohydrates, minerals, and trace elements generally required for microbial growth (2,7,8), it is not surprising that the fungus develops in this region of the kernel. However, significant variation has been observed in the incidence of A. flavus-infected kernels in contaminated lots of corn (6,14). In addition, the presence ofA. flavus in a commodity is not always associated with aflatoxin contamination (5). In laboratory studies differences in aflatoxin production by A. flavus have been attributed to heterogeneity of the fungal substrate (5). Synthetic media have rountinely supported minimal toxin production (1 to 60 mg of B1 per kg of medium), whereas maximum yields (700 to 900 mg of B1 per kg) occur on such commodities as autoclaved wheat, rice, cottonseed, and corn (5). Schroeder (13) found that a constituent(s) present in corn steep liquor increased the production of aflatoxin by Aspergillus parasiticus in a Czapek's broth medium. Studies by Mateles and Adye (1,9) of toxin production in a glucose-ammonia-salts synthetic medium showed that deletion of zinc (2 jg per g) reduced aflatoxin yield without restricting growth of the fungus. Therefore, Schroeder (13) proposed that the enhanced level of toxin detected in media enriched with corn steep liquor was the result of 763 zinc in the corn extract. Davis et al. (4) examined the effect of trace elements on A. flavus synthesis of aflatoxin in a sucrose-nitrate-salts medium. They found that zinc (5 fg per g), iron (2 ,g per g), and magnesium (100 Aig per g) were required for optimal toxin yield but that manganese levels of 1 Ag per g interfered slightly with toxin production. In a similar synthetic medium Reddy et al. (12) corroborated the requirement for zinc to achieve maximum aflatoxin elaboration but no increase was observed with added iron; omission of manganese from the medium increased toxin accumulation. Trace elements in corn occur predominantly in the germ fraction (7,10). However, phytate (inositol hexaphosphate) in the germ strongly binds several elements, particularly zinc; the bound elements are not readily available biologieally (10,11). The ratio of phytate to metal ion levels in the corn germ and binding of trace elements by other constituents of the germ would be important factors in microelement availability. We compared the levels of trace elements in corn germ fractions from kernels naturally infected with A. flavus to germ from kernels free of the fungus. We also studied the variability in aflatoxin production by strains of Aspergillus spp. on autoclaved corn and by A. flavus on autoclaved, defatted corn germ amended with trace elements; by determining toxin yields we measured availability of trace elements to the fungus. This investigation was presented at the May 12-17 annual meeting of the American Society for Microbiology in Chicago, Ill., 1974. MATERIALS AND METHODS Samples of whole corn, acquired from commercial dealers, were either heavily infected with A. flavus and contaminated with aflatoxin or the kernels were free of both fungus and toxin. A. flavus in the test corn was detected by surface-sterilization of kernels with 1% sodium hypochlorite for 1 min, washing the corn with sterile water, and placing the seed on ME agar (malt extract, 30 g, and agar, 15 g per liter). Petri plates were incubated for 3 days at 28 C, and fungi on the kernels were identified under a mocroscope. Corn fractions were obtained either from commercial sources or by hand dissection. Composition of similar commercial and hand-excised corn fractions has been reported (2,7,8). In this study manual excision of corn germ was carried out on kernels that had been steeped in water for 10 min before removal of the pericarp and subsequent dissection with special precaution to avoid contamination of the germ with other kernel constituents. Germ fractions for trace element analyses came from both A. flavus-infected and A. flavus-free corn samples. Kernels exhibiting bright greenish-yellow (BGY) fluorescence under ultraviolet light (14) were selected for germ excision; 99% of the BGY-positive kernels were infected with A. flavus. Elemental analyses were carried out by flame atomic absorption; whole kernel corn and hand-dissected germ were wet ashed with nitric acid and assayed by the procedure described by Garcia, Blessin, and Inglett (Cereal Chem., in press). Strains of A. parasiticus NRRL 2999, 5013, and 5862 and A. flavus NRRL 3251, 3357, and 5520 were supplied by the ARS Culture Collection maintained at the Northern Regional Research Laboratory. Cultures were grown on potato dextrose agar slants, and spores for inocula acquired from the slants were incubated for 10 days at 28 C. Aflatoxin production studies were carried out with 1-g samples of whole corn and commercial corn fractions in 50-ml Erlenmeyer flasks with addition of 1 ml of sterile water or the appropriate solution of a specific trace element. Test flasks were autoclaved for 20 min at 121 C and the sterile substrates were inoculated with 0.5 ml of spore suspension (7.0 x 107 spores per ml). Aqueous solutions of the following compounds were used as trace element additives: ZnSO,.7H20; CuSO4-5H20; CrO2; Pb(C2HO2)2. 3H20; CdCl2 .2H20; FeCl1; and MnSO4.H20. Inoculated flasks were statically incubated at 25 C. Contents of individual flasks were extracted with 250 ml of chloroform in a Waring blender for 3 min. The extraction solvent was recovered by filtration with subsequent removal of water by addition of anhydrous sodium sulfate; the chloroform solution was vacuumevaporated to the desired volume. Fractions of test solutions were spotted on thin-layer (TLC) plates coated with 0.5-mm Adsorbisil-1 (Applied Science Laboratories Inc., State College, Pa.). The plates were individually developed in ether (3) and subsequently in chloroform:acetone:water (92:8:1). Quantitative determinations were made on developed TLC plates by comparison of fluorescent intensities of alfatoxin from the fermentation extracts with reference standards of pure toxin. RESULTS AND DISCUSSION Trace element levels in corn naturally infected with A. flavus. In preliminary studies we examined the relationship between trace element concentrations in corn and the natural occurrence of A. flavus. Analyses were carried out on whole kernel corn and on manually excised corn germ from samples that were either infected with A. flavus or free of the fungus ( Table 1). In addition to trace elements, the levels of phosphorus in test corn were also determined to provide information on phytate concentration. About 90% of the phosphorus in corn germ occurs as phytate (10). Generally, element levels were similar or higher in whole kernels of A. flavus-infected corn than in kernels free of the fungus. However, chromium concentrations were an exception; A. flavus-free corn had a distinctly higher level of the element than kernels infected by the fungus. Since the chromium levels in the A. flavus-free whole kernel and germ fractions were approximately equal, it was concluded that the endospermhull component of corn in this sample contained significant quantities of the metal. A pattern of increased metal levels was observed in fungalinfected germ compared with a germ fraction free of the fungus; the greatest differences (40 to a Whole kernel values represent means of four independent 10-g samples; full-fat germ values were acquired from single determinations. A. flavus was observed in 75% of the infected kernels, and the fungus was not detected in A. flavus-free corn. " Levels in milligrams of corn (dry weight) per gram. 60%) were observed in manganese, cadmium, and chromium concentrations. Although the data in Table 1 suggest a correlation between enhanced trace element levels in corn germ and the presence of A. flavus, the increased phytate concentration in the fungal-infected germ indicates that more of the elements in this fraction would be biologically unavailable. Therefore, a clear cause-effect relationship between trace element levels and natural infection of corn by A. flavus cannot be established by these results. Aflatoxin production on corn. Six A. parasiticus and A. flavus strains were tested for toxin yields on a ground, whole corn substrate that was initially toxin free. Two of the three strains of A. parasiticus produced more aflatoxin B, after 7 days of static incubation at 25 C than did the A. flavus isolates ( Aflatoxin production by A. flavus on autoclaved, ground whole corn was compared with yields on other autoclaved corn fractions (Fig. 1). During the 5-day fermentation, toxin elaboration on whole corn and commercial, full-fat germ fractions was about equal. Corn endo- sperm (grits) supported toxin production comparable to whole corn and full-fat germ during the initial 3 days of incubation; after 5 days, aflatoxin yield on endosperm was 22% below the optimal levels observed in whole corn and full-fat germ fractions. Defatted germ was the poorest substrate for toxin production; the yield after 5 days was 75% below the maximum accumulation observed on both ground, whole corn and full-fat germ. Availability of trace elements in corn germ. Differences between trace element levels in germ fractions from either A. flavus-infected or A. flavus-free corn, detected in preliminary studies, indicated a possible relationship between the concentration of metals in the germ and natural occurrence of the fungus. The interaction between trace elements and A. flavus development was further examined by testing the influence of added trace metals on aflatoxin synthesis on an autoclaved sample of commercial, defatted corn germ. Corn germ was selected for the trace element studies because metals occur predominantly in this fraction of the kernel; defatted germ was chosen because the reduced rate of aflatoxin synthesis on this substrate provided a more sensitive medium for observing subtle changes in toxin yields. Binding of trace elements by phytate in corn germ would interfere with biological availability of trace metals (8,10,11). Aflatoxin yields were plotted on defatted corn germ with zinc and iron added at levels ranging from 50 to 1,000 gg per g of germ (Fig. 2). Addition of 250 ug of zinc per g of germ 765 VOL. 28,1974 increased aflatoxin yield 2.5 times after 3 days of incubation at 25 C; maximum production of the toxin was observed at 250 to 500 /ig of zinc per g of germ. Since previous studies had shown that addition of 1 to 5 ,ug of zinc per ml of a submerged fermentation medium distinctly stimulated aflatoxin production (1,4,19), evidently the zinc in the corn germ (208 Mg per g) is not readily available for A. flavus biosynthesis because added quantities of the metal significantly increased toxin yields. Addition of iron to the corn germ substrate had no effect on toxin production. The effect of added manganese and copper (1 to 500 Ag per g) on aflatoxin production in defatted corn germ is plotted in Fig. 3. important factor in A. flavus production of aflatoxin. Although previous studies have shown that yields of aflatoxin in submerged fermentation can be restricted by low levels of manganese (1 ttg per g) (4), the synthesis of toxin on defatted germ was increased by the addition of 10 Mtg of the metal per g with no significant inhibition after addition of 10 to 500 Mg of the substance per g of germ. Clearly, the response of the toxin-producing fungus to manganese differs in development on defatted corn germ versus liquid media. Aflatoxin production on defatted corn germ enriched with cadmium, chromium, or lead (levels ranged from 1 to 50 tig per g) is presented in Fig. 4. From 1 to 10 Mg of cadmium per g of germ increased toxin yield 2.1 times relative to unamended controls; 25 to 50 Mg of cadmium per g decreased aflatoxin accumulation to control levels. Low levels of chromium (1 Mg per g) also increased toxin yields about two times with only a slight reduction from maximum accumulation at 50 Mg per g. Low levels of lead (1 to 5 Ag per g) did not significantly increase toxin levels, but addition of 25 to 50 ,ug per g of the metal increased production. It appears that cadmium, chromium, and lead levels in defatted germ are not available at the concentrations required for optimal toxin synthesis. The inhibition of aflatoxin production by increased levels of cadmium resembled the effect observed with elevated concentrations of copper. Studies of aflatoxin production on defatted corn germ enriched with trace elements demonstrate a relationship between addition of several metals and significant differences in toxin yields. Our results provide preliminary information on the association between a factor in corn, trace elements, and the process of A. flavus FIG. 4. Production of aflatoxin by A. flavus on defatted corn germ enriched with cadmium, lead, or chromium incubated statically for 3 days at 25 C. Toxin production was carried out on 1-g samples of germ in 50-ml Erlenmeyer flasks. Values represent means of triplicate test flasks assayed independently. infection and subsequent elaboration of aflatoxin.

v3-fos

2020-12-10T09:04:22.874Z

1974-09-01T00:00:00.000Z

237232821

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Studies on the Cecal Microflora of Commercial Broiler Chickens A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO2 and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes. A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO2 and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes. There is little information available describing the predominant kinds of bacteria occurring in the intestinal tract of the commercial broiler. Studies on layer and broiler chickens have shown that the cecum contains the largest number of bacteria, most of which are strict anaerobes (2,4,18,22,23). Barnes and Impey (2,3) have isolated from poultry ceca (chickens, turkeys, pheasants, and ducks) several groups of anaerobic streptococci as well as gram-negative and gram-positive nonsporeforming anaerobes including species of Bacteroides, Fusobacterium, Eubacterium, Propionibacterium, and Bifidobacterium. In the few remaining studies concerned with chicken microflora, characterization of the cecal bacteria was limited to only a few groups which were cultured on selective media. In contrast, we previously observed that using nonselective rumen fluid media and strict anaerobic methods devised for rumen bacteria (20) allowed a large percentage of anaerobes to be isolated from the cecum of "laboratoryreared" broilers (5-week-old). Ninety percent of the microflora cultured in this prior study was composed of facultatively anaerobic cocci and streptococci and strictly anaerobic species of Peptostreptococcus, Propionibacterium, Eubacterium, Clostridium, Bacteroides, and unidentified gram-negative organisms. The work reported here extends our initial studies on the major groups of bacteria isolated from the chicken to include the cecal microflora of broilers which were reared under commercial husbandry conditions. Data on variability in microbial populations among samples and some growth requirements of the predominant anaerobes are also presented. MATERIALS AND METHODS Animals. Five-week-old cockerels (White Cornish cockerel x White Rock hen) maintained on as antibiotic-free grower ration (containing a coccidiostat) were obtained from a commercial broiler facility (Foster Poultry Frms, Livingston, Calif.). Cecal samples (3 to 4 g wet weight) were taken from animals (0.9 to 1.0 kg) which had been killed by CO2-asphyxiation. Culture methods and isolation of cecal bacteria. Direct microscopic counting, anaerobic culture techniques, sampling procedure, and nonselective anaerobic roll tube media used in this study were the same as 439 440 SALANITRO, BLAKE, AND MUIRHEAD previously described (20). Portions of 10-9 dilutions of the cecal samples were inoculated into roll tubes of M98-5 media. After 6 days of incubation at 37 C, 300 colonies were picked from roll tubes (50 to 60 colonies per tube) inoculated with cecal samples from six individual birds. The colonies were subcultured to slant media of similar composition (20). Mixed culture isolates were purified by restreaking on M98-5 media (15). Initial criteria used for grouping pure cultures of strains were morphology (phase contrast microscopy), Gram stain reaction, oxygen sensitivity (facultative anaerobes or strict anaerobes), growth bn glucose, and fermentation products formed from glucose. Media and methods used for these tests have been given (20). Carbohydrate fermentation, physiological tests, and identification of isolates. Tests to determine the ability of representative strains of anaerobes from bacterial groups in Table 1 to ferment several carbohydrates, to hydrolyze esculin and starch, to reduce nitrate, and to produce indole, H2, and H2S were performed. The basal media and methods used in these tests have also been described (20). Identification of strains was based on comparison of these various physiological features with those of known poultry isolates (2,3), classification schemes of Holdeman and Moore (15) and other published data on anaerobes. Analytical method for fermentation acids. Fermentation acids (formic, acetic, propionic, butyric, APPL. MICROBIOL. lactic, and succinic) elaborated in glucose-containing media were analyzed by a gas chromatographic procedure adapted from the method of Lambert and Moss (16) for the preparation of butyl esters. An estimate of the amount of each acid formed was determined from standard curves of authentic butyl esters (obtained from Pfaltz & Bauer, Inc.). The gas chromatograph used was a Hewlett-Packard model 5754B equipped with flame ionization detector an digital integrator. Ethanol in culture media was determined enzymatically with alcohol dehydrogenase (Sigma Kit no. 331-UV). Growth characteristics of the various strains were determined in media containing basal medium plus added components. The basal medium consisted of mineral solutions 1 and 2, glucose, Na2CO%, cysteinehydrochloride, and NaS at the same concentrations as given for IM. The following constituents were included to give the final concentrations (wt/vol, vol/vol, or mM): hemin (0.0002%), Trypticase (0.2%), yeast extract (0.2%), rumen fluid (CRF 2, 20%), volatile fatty acid mixture (29.5 mM acetic, 8 RESULTS AND DISCUSSION Isolation of cecal bacteria from broiler chickens. Initially, 300 anaerobic strains were picked. Some morphologically heterogeneous isolates were then reisolated, thus increasing the total number of strains examined to 325. The percent of the total microflora (direct microscopic counts) cultured in M98-5 medium varied among the six samples from 49.3 to 80.9% (mean of 59.6%). A comparison of different media for the isolation of cecal anaerobes from commercial broilers was part of a previous study (20). Strains were tentatively classified on the basis of morphology, carbohydrate fermentation, fermentation products, and other physiological and biochemical features (Tables 1 and 2). A description of the predominant groups of cecal bacteria isolated follows. With the exception of two groups of facultatively anaerobic cocci and rods (group II and VII, Table 1), the majority of these bacteria were gram-positive and strict anaerobes. Facultatively anaerobic bacteria. Two types of facultative anaerobes were isolated: gram-positive cocci (group II) and gram-negative, motile rods (group VII) comprising 12.6 and 4.9% of the total isolated microflora, respectively. The gram-positive cocci, which were also isolated from chicken cecal contents in a previous study (20), produce lactic acid as a major fermentation product. Under anaerobic conditions they form acid from glucose, fructose, lactose, maltose, and sucrose. All of the grampositive cocci examined (eight strains) hydrolyze gelatin and tributyrin and reduce nitrate to nitrite. Catalase activity could be demonstrated in cultures grown aerobically on plate media but not in cultures grown anaerobically in prereduced media (20). Although these organisms were not identified previously (20), we now believe they may be related to the genus Staphylococcus. Tentative identification is based on the fact that they are facultatively anaerobic and produce acid from glucose anaerobically and aerobically (1). In addition, they produce major amounts of lactic acid from glucose (anaerobically) as do other strains of Staphylococcus (S. aureus and S. epidermidis) we have examined. Facultatively anaerobic, gram-negative rods of group VII were presumptively identified as Escherichia coli in tests with the improved Enterotube (Roche Diagnostics). These bacteria were present in five of the six cecal samples (Table 3) and constituted 2 to 13% of the isolated strains. Axnaerobic cocci. Bacteria in group I were gram-negative, pleomorphic cells with many club, dumbbell and budding forms in pairs and streptococcal-like chains. It is uncertain whether these spherical to elongated cells are truly cocci or rods because of their extreme pleomorphism. We have temporarily designated them as budding cocci. Strains similar to this unnamed species have been isolated from the chicken cecum (4,20), human feces (J. Gossling, Abstr. Annu. Meet. Amer. Soc. Microbiol., p. 81, 1972) and human uterus (12). In a rumen fluid basal medium these bacteria do not ferment sugars but do hydrolyze esculin ( Table 2). Fermentation products from glucose include minor amounts of formic, acetic, and butyric acids and some H2 gas (0 to 2.6%, vol/vol). Strains in group III are large (1.5 to 2.0 tm) gram-positive cocci found in pairs and chains which are probably related to species of Peptostreptococcus Kluyver and Van Niel (19). These anaerobic streptococci differed from Peptostreptococci cultured from chicken cecal contents in a previous study (20) inasmuch as they fermented several sugars. These strains resembled P. intermedius as described by Holdeman and Moore (15) except for the production of indole and hydrolysis of esculin. Group III 441 VOL. 28, 1974 SALANITRO, BLAKE, AND MUIRHEAD a Media and methods for most tests are given in references. Production of ammonia was determined with Nessler reagent in media used for indole production. Lipolysis was determined in basal media containing 1% (vol/vol) tributyrin (Sigma Chemical Co.) as fermentable substrate. Reactions given are for most strains without a group. Symbols: -, no fermentation (terminal pH 6.3 to 7.0) or no reaction; a, acid reaction (terminal pH 5.5 or less); w, weak reaction (terminal pH 5.6 to 6.2); +, positive reaction; V, variable reaction. Superscripts refer to reactions of a few strains. None of the strains fermented amygdalin or inositol and only 1% of all isolates fermented melezitose. Glycogen and rhamnose were fermented only by group Vc. Motile strains were observed in group VIb. b Designation of amounts of fermentation products is similar to that given in the footnote of Table 1. Products in parentheses are formed by a few strains. strains constituted a small part of the total microflora (Table 1) and were observed in only two out of six cecal samples. Anaerobic gram-positive rods. As in a previous study (20), this group of gram-positive, nonsporeforming rods represented the largest portion of the cecal bacteria isolated. Half of the strains in group IV (18%) were identified as Propionibacterium acnes (IVa). These belonged to serotypes I and II on the basis of sorbitol fermentation (15). P. acnes was also isolated from poultry cecal material by Barnes and Impey (3). Groups IVb and IVc resembled species of Eubacterium as they were anaerobic, gram-positive rods primarily producing lactic and butyric acids from glucose (15). Group IVb strains were oval-shaped cells or rods with known Eubacterium species (15). Group IVc included short and long slender rods (0.5 to 1.0 by 2.5 to 5.0 Mm) in chains. Although these strains appear to be closely related to E. tortuosum described by Holdeman and Moore (15), most of our strains ferment salicin and produce indole. Bacteria in group IVd were gram-positive to gram-variable rods (1.0 by 2.5 to 5.0 um) with rounded ends found in long chains. These bacteria differed from any previously described nonsporeforming, gram-positive anaerobes as they characteristically produced acetic and succinic acids and H2 gas from glucose. They may be related to species of Eubacterium on the basis of presumptive criteria for this genus (15). Similar strains of grampositive, nonsporeforming, succinate-producing bacteria have been isolated by E. M. Barnes from poultry cecal material (personal communication). Anaerobic, gram-negative rods. Group V was composed of gram-negative rods and totaled 18.6% of the isolated microflora. Strains in group Va were variable with respect to the fermentation tests given in Table 2. Representative strains were short and long fusiform-shaped rods (1.5 to 2.5 by 2.5 to 5.0 Mm) as singles, pairs, and chains. Fermentation of fructose, glucose, maltose, sucrose, and trehalose as well as formation of formic and acetic acids as major products from glucose and pyruvate were some predominant characteristics of the strains tested. Morphological features of these organisms and their produsts from glucose were similar to Bacteroides biacutus and Bacteroides clostridiiformis (15). They differed from B. biacutus in that melezitose and lactose were not fermented. Phase contrast microscopy revealed that a few strains appeared to have small spores or vacuoles located centrally and subterminally. It is not known whether these are true spores since none of the strains survived a spore heat test (15). Identification of this group is provisional until characteristics of additional strains are examined. Strains similar to group Va were isolated and identified as B. clostridiiformis from chicken cecal contents (20) and from poultry ceca (2). Group Vb were large, blunt-ended rods (2.0 by 5.0 to 7.5 Mm) occurring mainly as singles and pairs. These species were identified as Bacteroides hypermegas since many fermentation reactions agreed well with those of Holdeman and Moore (15). Our organisms fermented a wide variety of sugars, hydrolyzed tribytyrin, and produced major amounts of propionic and acetic acids from glucose. Strains of B. hypermegas were first described by Harrison and Hansen (14) and later isolated by Goldberg et al. (13) and Barnes and Impey (2) from poultry ceca. Bacteria in Proup Vc appear to be similar to Bacteroides fragilis as described by Holdeman and Moore (15). These gram-negative rods (measuring 0.5 to 1.0 by 1.5 to 3.0 Mm) produced succinic and acetic acids and H2 gas (2.1 to 4.8%, vol/vol) from glucose fermentation. All four isolates we examined differed from known B. fragilis strains in that our isolates converted threonine to propionate. They differed from B. ruminicola in that H2 gas was produced in the culture media (5). Preliminary studies by M. P. Bryant have indicated that most strains of B. fragilis can be distinguished from B. ruminicola on the basis of H2 production (unpublished observations). It is possible, therefore, that the succinate-producing, gram-negative rods isolated in this study may be variants of B. fragilis which can convert threonine to propionate. Barnes and Impey (2) have also isolated B. fragilis from poultry ceca. Anaerobic, sporeforming rods. Group VI comprised 16% of the isolated cecal microflora and consisted of three types of Clostridium species which did not liquefy gelatin. Group VIa was represented by large, nonmotile rods measuring 1.5 to 2.5 by 5.0 Mm (some as long as 20 Mum) with blunt ends and subterminal spores. The two strains tested produced acid from fructose, glucose, and mannose and formed butyric and lactic acids as well as copious amounts of H2 gas (9 to 13%, vol/vol) from glucose. These strains may be similar to C. tyrobutyricum on the basis of sugars fermented, products from glucose (15), and lactate fermentation (7). However, our strains appeared to be nonmotile. Strains in group VIb were pleomorphic, motile rods (1.0 by 2.5 to 5.0,um) with terminal and subterminal spores; some cells were typically fusiform-and spindle-shaped arranged as singles, pairs, and a few chains. These organisms weakly fermented fructose and glucose and formed primarily butyric acid from glucose. These strains were similar to those isolated in a previous study (20). Group VIc consisted of oval-shaped rods (1.5 to 2.0 by 2.5 to 5.0 Mm) with subterminal spores occurring as singles, pairs, or chains. These bacteria were nonreactive in several tests (Table 2), producing minor amounts of butyric acid and H2 gas from glucose but large amounts of butyric acid from lactate fermentation. None of the organisms in groups VIb and VIc could be identified as being similar to known species of Clostridium described by Holdeman Preliminary findings on the growth requirements of anaerobic strains. Our previous study on cecal bacteria (20) indicated that rumen fluid and yeast extract stimulated the growth of many anaerobes. Since the growth requirements of chicken intestinal bacteria are largely unknown, we attempted a survey of some factors which might enhance the growth of several cecal isolates from the various morphological groups (Table 1). These data are shown in Table 3. Preliminary experiments (data not shown) with culture gas phase indicated that good growth was observed with Na2CO,-containing media prepared under CO2 or C021N2 atmosphere. Maximun culture density for most strains was achieved with a medium containing Na2CO,-CO2 buffer. Carbon dioxide may be stimulatory to the growth of most chicken cecal anaerobes; however, more rigorous experiments are needed to establish whether particular species of cecal bacteria have an absolute requirement for CO2. Work by Dehority (11) with ruminal anaerobes has revealed that many strains required or were stimulated by CO2. A simple basal medium consisting of minerals, glucose, Na2CO,-CO2, and cysteine-sulfide did not support the growth of any cecal strain examined. The addition of Trypticase and hemin, in particular, to.this medium stimulated the growth of strains similar to Bacteroides fragilis (group Vc). The growth stimulatory properties of hemin for intestinal anaerobes are well known and have been described for human intestinal isolates of B. fragilis (15,17) and ruminal strains of Bacteroides ruminicola (9,10). When yeast extract was included in the basal medium with Trypticase and hemin, the growth of strains from 8 of 11 groups was enhanced. When a vitamin mixture was added to the basal medium, only strains in group Vb grew (Table 3) as well as one strain each from groups Va and VIa (data not shown). Addition of rumen fluid (20% vol/vol) alone to the basal medium supported the growth of only a few groups (Va, Vb, and VIa). Components in rumen fluid other than volatile fatty acids and hemin appear to be required or highly stimulatory for strains in group I (gram-negative, pleomorphic cocci). Most strains of the various groups of cecal bacteria grew well in basal media supplemented with rumen fluid, Trypticase, hemin, and yeast extract (or a vitamin mixture in place of yeast extract). Hemin could be deleted from media containing rumen fluid without affecting the growth of hemin-stimulated strains (group Vc). In this respect, Caldwell et al. (10) showed that rumen fluid contains heme. Although a mixture of volatile fatty acids (VFA) could partially reproduce the stimulation of groups IVc and Va brought about by rumen fluid, VFA may not be required for the growth of most cecal anaerobes. Liver or cecal extract added to the basal medium only enhanced the growth of group Vb strains (B. hypermegas). These observations are consistent with our previous findings that most chicken cecal bacteria do not require liver or cecal extract for growth (20). The growth of some strains of chicken cecal anaerobes isolated by E. M. Barnes, however, appears to be enhanced by a factor(s) in liver extract (unpublished observations). The data in Table 3 suggest that most anaerobic cecal bacteria could be grown in a Na2CO,-CO2 buffered medium containing minerals, cysteine-sulfide (as reducing agent), carbohydrate energy source, rumen fluid, Trypticase, and yeast extract. The fact that yeast extract or a vitamin mixture is highly stimulatory to many cecal bacteria suggests that chicken intestinal anaerobes may have specific vitamin requirements for growth. The growth stimulation by vitamins such as p-aminobenzoic acid, biotin, folic acid, and vitamin B,2 for the cellulolytic rumen anaerobes, Ruminococcus species, and Bacteroides succinogenes (8,21), has been established. Not much is known about the vitamin requirements of noncellulolytic anaerobes. However, Varel and Bryant (24) have recently observed a B,2 requirement (replaced by methionine) for growth with human isolates of B. fragilis. Compositional variations in the cecal microflora of broilers. In this study (Table 1), 83% of the total isolated microflora (325 strains) of 5-week-old broilers consisted of (1) grampositive, facultative cocci (group II, 12.6%), (2) Propionibacterium acnes and Eubacterium sp. (group IV, 36.1%), (3) Bacteroides clostridiiformis, B. hypermegas, and strains similar to B. fragilis (group V, 18.6%) and (4) Clostridium sp. (group VI, 15.7%). The remaining groups of the flora were made up of gram-negative, budding cocci (group I, 5.2%), Peptostreptococcus (group III, 1.5%) and E. coli (group VII, 4.9%). Table 4 shows the distribution of the various morphological groups and subgroups isolated and indicates that considerable variation exists among individual cecal samples. The cecal profile of microorganisms from each animal suggests that some species are isolated with (or associated with) certain other species. For example, in samples 1, 2, and 3, the lactic acid-producing, facultatively anaerobic cocci (group II) were isolated with large numbers of other gram-positive organisms (primarily group IVa strains) VOL. 28,1974 along with relatively smaller numbers of sporeforming rods (group VI) and gram-negative rods (group V). In samples 4, 5, and 6, however, the gram-negative rods in group V were isolated with gram-positive rods (groups IVb, c, and d) and sporeforming rods (group VI). It is interesting to note that B. hypermegas (Vb) and B. fragilis-like strains (Vc) were isolated in high numbers (23.0 and 18.2% of the isolated microflora, respectively) in only two samples (4 and 5). These two species were not isolated in our initial study (20). Results of the previous study (20) on chicken cecal microflora of 5-week-old, "laboratoryreared" birds indicated that species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus sp., Propionibacterium acnes, Eubacterium sp., B. clostridiiformis, and Clostridium sp. were isolated. The types of anaerobes isolated from cecal material in our two studies were similar to those described by Barnes and Impey (2) from poultry ceca. However, the relative proportions of the various bacterial types differed. Few studies dealing with intestinal microflora in nonruminants consider the animal to animal variation when describing the predominant bacterial species isolated from intestinal material. Our data indicate that appreciable variation may exist in the cecal microflora among differ-ent animals (as well as within the same animal) reared under similar growth conditions (environment, food, water). These observations suggest that other factors (host-microbe or microbe-microbe interactions) may have some effect on the occurrence and distribution of the various bacterial species in the cecum. These must be taken into consideration in any study involving the isolation, cultivation, identification, and relative importance of species in a mixed culture habitat such as the chicken cecum.

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2020-12-10T09:06:54.746Z

1974-02-01T00:00:00.000Z

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Polygalacturonate Lyase Production by Bacillus subtilis and Flavobacterium pectinovorum Nutritional factors relating to the production of polygalacturonate lyases by strains of Bacillus subtilis and Flavobacterium pectinovorum were examined. Studies were carried out in shake flask cultures. In the case of B. subtilis the enzyme was produced constitutively, whereas in the case of F. pectinovorum it was only produced in quantity in the presence of pectic substances. Glucose was the most suitable carbon source for production of the polygalacturonate lyase of B. subtilis; of the nitrogen sources examined, the highest activities per milliliter of supernatant and per milligram of cells were obtained with glutamine and ammonium sulfate, respectively. The pattern of enzyme production and growth was similar although enzyme production ceased at pH 5.3. Sodium polypectate was the best inducer of polygalacturonate lyase with F. pectinovorum. Highest activity per milliliter of cell-free supernatant was obtained with skin milk powder as nitrogen source, although ammonium sulfate gave highest enzyme production per unit of biomass. Growth of F. pectinovorum occurred between pH 5.7 and 7.2. Enzyme production occurred during active growth and was independent of the pH of the medium. Many species of wood are naturally resistant to treatment with preservatives, even when applied under pressure. During storage of Sitka spruce (Picea sitchensis) in water, bacterial degradation of the tori and bordered pit membranes takes place and this results in a marked increase in the permeability of the wood (1, 2). A number of bacterial species have been isolated from the sap of Sitka spruce poles that had been stored under water; two of these isolates were shown to possess high pectinolytic enzyme activity (4). This observation was significant because the pit membranes in the sapwood contain much pectic material. When these isolates were inoculated into sapwood blocks under laboratory conditions an increase in the permeability of the blocks occurred (3). The bacterial isolates have been identified as strains of Bacillus subtilis and Flavobacterium pectinovorum. Both isolates elaborate polygalacturonate lyase extracellularly and grow well and produce this enzyme in sapwood blocks in laboratory experiments. Polygalacturonate lyase activity has also been detected in sap, expressed from water-stored Sitka spruce (18). Polygalacturonate lyase was the only pectic enzyme produced by the bacterial isolates and was likewise the only pectinase detectable in expressed sap of water-stored Sitka spruce. The importance of pectinases in the process is indicated by the observation that a commercial pectinase was also capable of increasing the permeability of sapwood blocks (3). This investigation is a comparative investigation of some factors affecting production of polygalacturonate lyase by the isolates in batch culture. MATERIALS AND METHODS The basal mineral medium consisted of, in grams per liter: K2HPO4, 5.0 g; KH2PO4, 1.0 g; KCl, 1.0 g; MgCl,.6H20, 0.2 g; CaCl2 2H20, 0.1 g; MnSo4-4H20, 0.001 g; FeSO4.7H,0, 0.0005 g. Carbon sources, 0.5% (wt/vol), were added to this medium. The nitrogen sources consisted of complex nitrogen, (peptone, casein, etc.), 1.0% (wt/vol), amino acids 0.5% (wt/vol) or inorganic N, 0.2% (wt/vol). In studies on the effects of carbon and nitrogen sources on enzyme production, the initial pH (pHi) of the medium was 7.0 to 7.2. Media were dispensed in 50-ml volumes in 250-ml Erlenmeyer flasks, and autoclaved at 121 C for 15 min. A standard inoculum was prepared as follows: actively growing cells were centrifuged from the basal mineral medium containing bacteriological peptone (0.5%, wt/vol), washed, and suspended in sterile saline to give an optical density (OD) reading of 10.0 in an EEL spectra colorimeter at 600 nm. All media were inoculated with 1.0 ml of this standard suspension and shaken at 150 rpm in a New Brunswick orbital incubator (model G25) set at 27 C. Enzyme activity was measured in the cell-free supernatant (CFS). In studying the effect of pH on growth and enzyme production, biomass and enzyme activity estimations were carried out throughout the growth cycle of the organism. In other investigations, enzyme activity was assayed when enzyme production had ceased in the stationary phase of growth. Variations in activity between duplicate culture flasks were negligible, and results were reproducible on repetition of experiments. Acid-soluble pectic acid (ASPA) was prepared by the method of McCready and Seegmiller (11). Polygalacturonate lyase activity was measured with a solution containing 0.2% (wt/vol) ASPA, 0.001 M CaCl2, and 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 8.0. The substrate was prepared immediately before use. Appropriately diluted, cell-free supernatant (0.1-ml samples) was added to 2.0 ml of substrate in all assays. The rate of change in absorbance at 235 nm was measured in a 1-cm cell, using a Pye Unicam SP 500 spectrophotometer equipped with a constant temperature unit. An enzyme unit is defined as the lyase activity releasing 1 Mmol of product per min at 30 C. A molar absorbtivity of 4,600 M-'/cm was used for this calculation (16). Activities are expressed in units per milliliter of CFS. However, to establish that differences in enzyme activities were not merely due to differences in bacterial growth, activity was also determined as a function of biomass (i.e., in units per milligram of biomass). This value provides a better measure of enzyme induction. Biomass was measured in OD units at 600 nm in an EEL spectrophotometer. OD units were converted to milligrams (dry weight) of cells by reference to a standard curve. Results of biomass determinations are expressed as milligrams per milliliter of CFS. When an insoluble carbon source was used in the growth medium, biomass was not measured. RESULTS Enzyme production by B. subtilis. The effect of carbon source on polygalacturonate lyase production by B. subtilis is summarized in Table 1. A 0.2% (wt/vol) amount of (NH4)2SO4 was added as nitrogen source to the basal medium. Although considerable enzyme activity was produced on many carbohydrates, glucose was clearly the best carbon substrate. Saccharides containing two or more glucose units were the poorest inducers. Glucose was incorporated as carbohydrate in media used to investigate the effect of nitrogen source on enzyme production. The results are presented in j'able 2. Although highest enzyme activity per milliliter of CFS was measured in media containing glutamine, highest induction as related to biomass was detected with (NH,)2SO as nitrogen source. However, significant levels of enzyme activity were measured with all nitrogen sources. The effect of pH on growth and polygalacturonate lyase production by the B. subtilis isolate is illustrated in Fig. 1. (NH4)2SO4 and glucose were incorporated into the basal salts medium. Although bacterial growth occurred between pH 4.7 to 7.5, the growth rate decreased with increase in pH. Because growth was accompanied by acid production, growth stopped when the pH dropped to about 4.5. Generally, the pattern for enzyme production was similar to the growth pattern. However, production stopped when the pH of the medium dropped to 5.3, although growth was not impaired. Enzyme production by F. pectinovorum. In determining the effect of carbon source on polygalacturonate lyase production by F. pectinovorum, (NHj2SO4 was added as nitrogen source to the basal medium. The results are presented in Table 3. The enzyme was only induced by pectic substances. On other carbohydrates only a very low level of activity was observed. Sodium polypectate was added as inducer to media used in investigating the effect of nitrogen source on enzyme elaboration by this organism (Table 4). While highest activity per milliliter of CFS was obtained when skim milk was used as a nitrogen source, activity reflected the high biomass production. The carbohydrate content of skim milk made little or no contribution to the level of activity produced (Table 4). (NH4)2SO4 caused highest enzyme production per unit of biomass. Apart from skim milk, enzyme production on complex nitrogen sources was completely inhibited or low ( Table 4). The influence of pH on growth and enzyme APPL. MICROBIOL. production is illustrated in Fig. 2. Sodium polypectate and (NH,)2SO were added to the basal medium in this experiment. Bacterial growth, which in this case was accompanied by a slight rise in pH, occurred between pH 5.7 and 7.2. Within this range the growth rate increased with increase in pH. Enzyme production occurred during active growth by the organism. Although enzyme production was affected by the growth rate, it appeared to be otherwise independent of the pH of the medium. DISCUSSION Although both organisms perform a similar function in increasing wood permeability, the above study reveals that the polygalacturonate lyase is constitutively produced by the B. subtilis isolate but is only induced in F. pectinovorum when the medium contains pectic substances. Apart from F. pectinovorum, inducible polygalacturonate lyase has been observed with Erwinia spp. (21), Pseudomonas fluorescens (5,7,20), Pseudomonas marginalis (13), xanthomonads (17), Xanthomonas campestris (14), and Clostridium multifermentans (9). On the other hand, although constitutive polygalacturonate lyase synthesis was observed with Aeromonas liquefaciens (8) and with Erwinia spp. (12), in contrast to B. subtilis, activity was repressed by glucose. Repression of constitutive enzyme production by glucose is a very common phenomenon (D. D. Brown and J. Monod, Fed. Proc., 20:222, 1961; 10). In a number of rumen bacteria (19) and in a strain of P. marginalis (21), constitutive polygalacturonate lyase production was not repressed by glucose. With both B. subtilis and F. pectinovorum, different degrees of repression were observed when nitrogen sources other than (NH,)2SO were added to the medium. In contrast to this, Hancock et al. (6) observed that polygalacturonate lyase elaboration by Hypomyces solani was reduced when individual amino acids or inorganic nitrogen sources were substituted for casein hydrolysates in the growth medium. As in the case of B. polymyxa, polygalacturonate lyases of F. pectinovorum and B. subtilis are produced mainly in the log phase of growth. The lyases of these two isolates are classical extracellular enzymes, according to the criteria established by Pollock (15), in that they are produced during active growth, are found in the cell-free supernatant, and are secreted before extensive cell lysis has occurred. The pH range for growth of B. subtilis was wider than that of F. pectinovorum. Enzyme production occurred in F. pectinovorum over its complete growth range, but not at lower pH values in the case of B. subtilis. Optimal elaboration of polygalacturonate lyase by H. solani (6) occurred within the pH range for production of this enzyme by the two isolates. During water storage, the pH of the sapwood generally varied from 7.0 to 5.0 (2). It has been illustrated that both organisms grow and elaborate the enzyme within this pH range. The growth rate of the B. subtilis isolate was higher at lower pH values, whereas F. pectinovorum had an increased growth rate at higher pH values. This study is part of a research project undertaken to investigate the possibility of utilizing artificial water storage tanks, seeded with selected bacteria, to increase permeability of softwoods to treatment with preservative. The high pectinolytic activity and resultant ability of the isolates to increase sapwood permeability renders them suitable for use in commercial storage plants for increasing wood penetrability of preservatives.

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1974-12-01T00:00:00.000Z

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Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 × 106 cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 107 or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected. taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected. In surveying sulfur metabolites in bacteria, an anaerobic species was desired which could be grown in a simple, defined medium with an inorganic sulfur source. Clostridium pasteurianum was selected since strain W-5 is widely used in biochemical studies and several defined media have been described for culture of this species (2-4, 7-9, 11). All of these media are based on salts, sucrose as energy source, biotin and p-aminobenzoic acid as cofactors with inorganic sulfate as the sulfur source; buffering is provided by phosphate or by supplements of CaCO, (2,3,8). We report here the relationship of growth of C. pasteurianum W-5 to sulfate concentration in a defined medium modified from that of Sargeant et al. (9). MATERIALS AND METHODS C. pasteurianum strain W-5 (no. 9486) was obtained from the National Collection of Industrial Bacteria. Reagents of analytical grade (Aristar) were used to prepare media in glass-distilled water. After investigating several media and methods of preparation, e.g., Carnahan and Castle (2), Carnahan et al. (3), and Lovenberg et al. (7), modifications of that of Sargeant et al. (9) were finally chosen. The best experi-I Authorized for publication as paper 4521 in the journal series of the Pennsylvania Agricultural Experiment Station. mental medium (DM 11) was prepared from stock solutions (Table 1) which were sterilized separately to decrease reaction between components: solutions A, B, and E by autoclaving, D by filtration, and C by allowing to stand. To avoid precipitation of hydrous iron oxide, stock solutions were combined as follows. Solution A was swirled and 0.1 ml of solution C was added. Then, solution E (1 ml) was added to make the sulfate concentration 0.6 mM, unless otherwise stated. Finally, all of solution B and 0.4 ml of solution D were added. Solutions C and E could be stored indefinitely, but solution D was prepared fresh every 2 weeks. Medium DM 11 and variations were not suitable for preservation of stock cultures because cold storage for a week led to erratic growth, but subculturing daily (Carnahan et al. [3]) or every 2 to 3 days was satisfactory. Stock cultures were maintained on the potato medium of Jensen and Spencer (6). Experimental cultures were grown in 75 ml of DM 11 in 125-ml Erlenmeyer flasks. Flasks had cotton plugs (wrapped in cheese cloth) each carrying a plugged Pasteur pipette for passage of gas to mix the cell suspension and displace 0.. Either N. or 95% Ns-5% CO2 was bubbled through each culture system to sweep out 0.. Media were inoculated and incubated at 30 C, and cultures were sampled with sterile pipettes. Cultures were always resparged to displace any 03 admitted during inoculation and sampling. Rapidly growing cultures of C. pasteurianum produced enough gas to make continuous sparging unnecessary for cultures in well plugged flasks or tubes. Growth was measured by turbidity (absorbance at 99.9 RESULTS AND DISCUSSION As preliminary experiments revealed that not all factors important to culture of C. pasteurianum in simple defined medium had been identified, study of culture conditions was essential before utilization of sulfate could be investigated. Major problems included choice of initial pH and the level and nature of the inoculum culture. Large inocula (5 to 10%) used by most workers (H. C. Winter, personal communication) interfered with study of sulfate metabolism by decreasing the specific activities of labeled compounds. Unfortunately, C. pasteurianum failed to grow regularly in previously described media (2,3,7,9) with 1% inocula. Therefore, the effect of pH was reexamined first. The medium of Carnahan et al. (3) has an initial pH of 6.6 to 6.9, and several other media (7,9) provide even higher initial pH values at 7.5, presumably to counteract the reported rapid pH decline during growth. Sargeant et al. (9) stated that the pH should not fall below 5.6, and, although continuous adjustment of pH was recommended (4,9) for large scale cultures, it is impractical in work of the present type. Media containing powdered CaCO, (2,8) effectively control pH but are unsuitable for turbidity or dry weight measurements and microscopic counts. A study of the effect of initial pH on culture ( Fig. 1) revealed that growth rate was significantly decreased by an initial pH of 5.5 and much depressed at pH 7.2, even though this latter value was lower than that recommended by some workers (9). Both lag period and generation time were affected by pH 7.2; growth was optimal over the pH range of 5.8 to 6.5. pH also influenced the final yield of cells with the maximum for an initial pH of 6.2 to 6.5 in 0.1 M phosphate buffer (Fig. 2). The total yield could be increased further by adjustment of buffering capacity. For example, 0.2 M phosphate produced 10% more cells, and addition of solid CaCO, to 0.1 M phosphate increased the in this type of experiment occurred during exponential growth in 0.1 M phosphate in the pH range 5.5 to 5.6. Contrary to a report (9) that this organism does not grow below pH 5.2, continuing growth was repeatedly observed until the pH declined to 4 or a little below. It became evident during these studies that the nature of the inoculum played a major role in culture of C. pasteurianum. Figure 3 illustrates this influence in terms of the age of cultures used as inocula. A culture in DM 11 just approaching stationary phase was maintained under 95% N,-5% CO,. Seven 4-ml samples were removed at intervals and used to inoculate 75-ml cultures of DM 11 which were incubated under 95% N.-5% CO. and subsequently used as inocula for the final cultures illustrated in Fig. 3. Of these seven, sample A was transferred from the still growing stock culture at a time designated zero and ultimately provided the oldest inoculum used in the last stage of the experiment; sample B was taken after 2h, C after4h, D at 6.5 h, E at9h, andF and G after 13 h to form cultures subsequently providing the youngest inocula. Incubation was ml. Under the best conditions devised, the generation time increased markedly from a fairly constant minimal value of about 85 min as the inoculum size was decreased from about 107 cells per ml (turbidity of 0.04, approximately 1% inoculation from a culture at absorbance of 5 at 650 nm to infinity with 106 cells per ml. The possibility that an unknown factor might initiate growth in still more dilute cultures of C. pasteurianum led to unsuccessful attempts to demonstrate growth-promoting activity. These attempts included: control of redox potential of the medium with thioglycolate, cysteine, or ascorbic acid; addition of biologically important metal ions (5); removal of oxygen by sparging with inert gases uinduer a varictv of rondition. 4. Growth of C. pasteurianum in medium DM final turbidity in absence of additional sulfate with varying sulfate concentration. A large inocu-had increased more than the factor of two ,n (6 x 107 cells per ml) was employed. recorded (1) for growth of Escherichia coli on CULTURE AND GROWTH OF C. PASTEURIANUM internal reserves alone. As in Fig. 4, growth at the highest sulfate concentrations passed a maximum and declined by 25%. No threshold value of sulfate concentration, below which growth did not occur, could be determined. This result is similar to that reported (1) in the much more sensitive experiments with E. coli which did not reveal a maintenance requirement for sulfur. Modified experimental approaches, such as successful culture with much smaller inocula or use of washed cells as inocula, will be necessary before this analysis with C. pasteuruanum can be made more sensitive. However, the present limited growth induced by small additions of sulfate will permit labeling at high specific radioactivity and isolation and identification of sulfur metabolites by methods used earlier (10).

v3-fos

2020-12-10T09:04:13.102Z

1974-04-01T00:00:00.000Z

237230462

s2

Comparative Denitrification of Selected Microorganisms in a Culture Medium and in Autoclaved Soil The denitrifying behavior of selected soil bacteria was compared in a culture solution and in soil that was sterilized by autoclaving. The essential characteristics concerning nitrate reduction and the formation of nitrogenous gases did not change significantly for most bacteria in the two environments. Bacteria whose denitrification product was nitrous oxide evolved the same gas both in soil and in a liquid system, whereas other bacteria formed only nitrogen gas. The validity of laboratory observations in relation to field studies in the domain of denitrification is discussed and evaluated. The denitrifying behavior of selected soil bacteria was compared in a culture solution and in soil that was sterilized by autoclaving. The essential characteristics concerning nitrate reduction and the formation of nitrogenous gases did not change significantly for most bacteria in the two environments. Bacteria whose denitrification product was nitrous oxide evolved the same gas both in soil and in a liquid system, whereas other bacteria formed only nitrogen gas. The validity of laboratory observations in relation to field studies in the domain of denitrification is discussed and evaluated. Extrapolation of results from pure microbial studies to the natural environment has often been questioned. However, due to the complexity of a natural ecosystem such as soil, laboratory studies serve as the only feasible method of monitoring or controlling all the inherent variables. For example, in the nitrogen cycle, it is difficult to determine if the denitrification process is partially obscured by nitrifying organisms or by non-biological factors. Therefore, it is desirable to design experiments that indicate what effect the introduction of new variables has on the microbial process under investigation. The basic knowledge of the denitrification process was reviewed extensively some years ago (6,7), and, although much research has been performed in the meantime, essentially no new findings have been reported. However, since denitrification can be undesirable from an agricultural viewpoint (2) or desirable in the removal of nitrate-nitrogen from the environment (8), the factors regulating this process need to be further clarified in order to utilize the potential of denitrifying microorganisms for practical purposes. In this paper, the denitrifying characteristics of selected bacteria in an artificial growth medium and in an autoclaved soil system were compared to clarify whether the change from a chemically defined liquid to the solid and complex structure of soil causes an essential change in denitrification patterns. I Paper no. 4408 in the Journal Series of the Pennsylvania Agricultural Experiment Station. MATERIALS AND METHODS Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis, from the culture collection of the Department of Microbiology at the Pennsylvania State University, and three soil isolates designated as isolates A, D, and H (5) were used. The bacteria were transferred from nitrate agar (Difco) to liquid Giltay medium and grown for 2 days at 30 C before they were used for inoculation of autoclaved soil or liquid media. Giltay medium (1), adjusted to a pH of 7.0 with NaOH, was used for both the stock culture solution and liquid media experiments. The soil used in these experiments was a Hagerstown silt loam soil, pH 7.0; the indigenous NO--N concentration was 75 uig/g of soil; organic carbon was 1.8%; and the sand, silt, and clay content was 8.5%, 63.4%, and 28.1%, respectively. Soil (20 g) was placed into incubation flasks, and 10 ml of distilled water containing 6 mg of NO--N was added. The soil was sterilized by autoclaving three times for 30 min over a 2-to 3-day period with at least a 12-h interval. The soil samples were inoculated with 1.0 ml of the selected stock culture. Liquid cultures containing 50 ml of Giltay medium were inoculated with one loop of bacterial culture. Anaerobic samples were incubated in 125-ml bottles sealed with a one-hole rubber stopper containing a septum for gas sampling (Applied Science Laboratories, Inc., State College, Pa.). Anaerobic conditions were attained by flushing the bottles with helium until all air was removed, as indicated by gas chromatographic analysis. Uninoculated control samples were assayed after different time periods to determine possible atmospheric contamination. Soil was incubated aerobically in 125-ml Erlenmeyer flasks closed with foam tube plugs. The flasks were placed in a closed incubation chamber, which was continuously flushed with filtered air. To minimize drying of the soil, a large pan of water was placed 674 in the chamber to increase the humidity. Aerobic liquid samples were incubated on a rotary incubation shaker revolving at 200 oscillations per min. All samples, aerobic and anaerobic, were incubated at 30 C for 3 days. The experiments were repeated two or three times, and several replicates were evaluated for each specific treatment. Gas samples were taken from the anaerobic bottles through the septum with a gas-tight syringe and injected into a gas chromatograph (Varian Aerograph, model 1820). Each sample was split equally into two parallel columns (3 mm outside diameter) of Porapak Q (600 cm; 50-80 mesh), which indicated peaks of CO2 and N20, and molecular sieve 5A (450 cm; 45-60 mesh), which separated 0, and N2. The columns were maintained at 50 C, and dual thermal conductivity cells at 200 C served as detectors. The carrier gas, helium, flowed at a rate of 40 ml/min, and the filament current was 200 mA for the Porapak Q and 150 mA for the molecular sieve 5A column. The quantity of gases was determined by the use of an integrator and calculated by comparisons with pure standard gas samples. Nitrate was measured with a nitrate electrode (Orion Research Inc., Cambridge, Mass.), and nitrite was determined calorimetrically by the a-naphthylamine-sulfanilic acid procedure (3). RESULTS After incubation for 3 days, it was apparent that the disappearance of nitrate was slower in the soil than in the liquid Giltay medium (Fig. 1), but the essential features of nitrate transformation by the various bacteria were not affected under the two conditions, with the exception of P. aeruginosa. Isolates A and D produced a considerable amount of nitrous oxide in the culture medium as well as in autoclaved soil, but the intermediary formation of nitrite could not be observed. On the other hand, S. marcescens and isolate H showed similar characteristics, although the marked formation of NO2in Giltay medium was not so apparent under soil conditions. The growth of P. aeruginosa was very rapid in the culture solution, as indicated by increasing turbidity. At the same time, practically all NO8which disappeared was recovered as N2. However, when the Pseudomonas species was cultivated in soil, the NO,was used more slowly, and, although N, was the predominant gas, it was also possible to detect NO2and some N20. B. subtilis was also included in these experiments, although this bacterium is normally not considered to be a denitrifier. It was found that, both in autoclaved soil and in the culture solution, some NO,was transformed, and it could be recovered as NO,and N2. Simultaneously with the release of nitroge- nous gases, the formation of CO, was observed, and the results of one representative experiment are shown in Table 1. Isolate H and S. marcescens, which accumulated a considerable amount of NO2in the culture solution, showed a strong production of CO2 when compared with the other bacteria, but the weaker NO.,--transforming ability in soil was complemented with a relatively smaller formation of CO2. This fact is especially noteworthy if it is compared with the bacteria that did not accumulate NO2in Giltay medium: their production of CO2 was more extensive in autoclaved soil than in the culture medium, although less NO,disap- The small amount of CO, formation found in the sterile control samples appears to be of non-biological origins, e.g., CO2 may be produced by decarboxylation of organic compounds or decomposition of free carbonates (9). When incubation occurred under aerobic conditions, there was practically no disappearance of NO,in soil inoculated with the various bacteria ( Table 2). Only isolate H produced a considerable amount of NO,-, but its origin was not further clarified. A strong decrease in NO,was observed when P. aeruginosa or isolate H was cultivated aerobically in the culture solution. Since gas sampling and analysis under aerobic conditions, in which a continuous exchange of air took place, were not sensitive enough for measurement of change in the content of nitrogenous gases, no clear conclusions in relation to this phenomenon could be drawn. However, it was very obvious that all other bacteria did not reduce NO3in soil or in culture solution if air or 02 was continuously exchanged by flushing or shaking of the incubation flasks. The complete inhibition of denitrification was demonstrated only under highly aerobic conditions. In an experiment in which isolate A was cultivated in a closed vessel that was flushed with pure oxygen, air, or helium prior to incubation, the denitrification was obviously related to the decrease of available oxygen (Table 3). The reduction of NO3and its use during the denitrification process were clearly indicated by the corresponding formation of N20. A similar result was obtained when isolate A was inoculated in sterilized soil. Since growth in soil was slower during 3 days of incubation, there was no or very little disappearance of nitrate in an oxygen or air environment, respectively, whereas, in a helium environment, the missing NO3could be recovered as N20. DISCUSSION Stimulated by the recent concern with environmental pollution, much interest has been shown in enhancing the denitrifying abilities of microorganisms and, thereby, reducing the danger of nitrate contamination of groundwater. To study the denitrification process and the major influencing factors, it is necessary to have well-controlled conditions as well as pure microbial cultures for obtaining conclusive results. However, since the conditions designed in the laboratory have to be quite different from those existing in the microenvironments within the soil, the application of these results is questionable. The comparison of the activity of denitrifying microorganisms in a liquid growth medium and after inoculation into sterilized soil is an attempt to determine whether incubation under various environmental conditions will significantly affect the denitrifying potential of certain microbes. Numerous bacterial species can denitrify in culture media, but there are no reliable data demonstrating how the same organisms behave in soil. It was even stated that the major part of denitrification in nature is performed by microbes that are different from those investigated essentially under laboratory conditions (10). Beijerinck and Minkman (4) found that the most numerous denitrifying organisms under average soil conditions were some Bacillus species, and, therefore, they assumed that these would also be the most important denitrifiers. Woldendorp (11) found that a change of the denitrifying population occurs after the addition of nitrate to soil: Bacillus species were originally the dominant denitrifiers, but gramnegative rods were mainly responsible for denitrification after incubation with nitrate. Woldendorp also compared pure cultures of gramnegative rods with Bacillus species and concluded that soil conditions are not favorable for denitrification by the latter type of organisms. In our investigation, it was shown that the characteristics of bacteria isolated from soil and laboratory cultures of denitrifiers did not show an essential change in their denitrifying behavior, with the exception of P. aeruginosa, whether observed in a culture medium or in autoclaved soil. Growth and also denitrification were slower during a similar time period in soil than in a liquid solution, and some intermediates of the nitrate reduction process appeared in different quantities in the two media. The increased accumulation of nitrite in the liquid medium of S. marcescens and of isolate H could be correlated with the faster reduction of nitrate and a subsequent inhibition in further transformation to nitrogen gas. There is no doubt that the different media and their influence on growth and other metabolic activities can change the speed of reactions and may have a secondary influence on the denitrifying patterns of the microbes, but the results of the reported experiments demonstrated that the features of selected denitrifiers are, for most bacteria, comparable in autoclaved soil and liquid media. Stotzky (10) emphasized that, since "soil is the most complex microbial habitat," the study of processes under consideration may require various levels of experimental complexities: it may be necessary to move back and forth between in vitro model systems and soil in situ to obtain knowledge of microbial activity in a soil ecosystem.

v3-fos

2019-03-19T13:13:01.542Z

1974-01-01T00:00:00.000Z

236894724

s2

Preparation and Storage of High-Titer Lactic Streptococcus Bacteriophages Various techniques were employed for preparation of high-titer bacteriophage lysates of Streptococcus lactis, S. cremoris, and S. diacetilactis strains. Infection of a 4-h host culture in litmus milk at 30 C yielded the highest titers (2 × 109 to 4 × 1011 plaque-forming units/ml) for most phages. Host infection in lactose-containing broth produced similar virus numbers only when 0.1 M tris(hydroxymethyl)aminomethane buffer stabilized the pH. The pH at the time of infection as well as the inoculum phage titer were critical in obtaining high titers. Optimum conditions for infection in broth were coupled with a polyethylene glycol concentration procedure to routinely produce milligram quantities of phage from 1 liter of lysate. Neutralization of whey lysates, as a means of storage, offered no survival advantage over unneutralized samples. Storage of phage lysates in a 15% glycerol whey solution at -22 C yielded a high rate of survival in most cases, even with repeated freezing and thawing, over a period of 24 months. Lactic streptococci are of critical importance to the dairy fermentation industry because these bacteria supply the lactic acid for curd production and their metabolic products impart characteristic and desirable flavors. Bacteriophage infection of these starter cultures results in insufficient acid production and usually a failure of the fermentation. The economic and public health consequences of these failures are well known (6). Various approaches have been utilized in an attempt to minimize bacteriophage infection during dairy fermentations. The use of culture rotation (4,6,15), mixed strain starter cultures (6,15,21), and a phage inhibitory medium (12) are currently in general use. These techniques never completely prevent failures, and constant precautions to prevent culture infections are advised. The use of mixed strains and starter rotation relies on the utilization of lactic streptococci that are resistant to a diversity of bacteriophage. Periodic examination of virus resistance patterns of starter cultures would be advisable. However, convenient techniques for the isolation and maintenance of bacteriophage stocks, which would be useful to industry, presently are not available. Previous studies of lactic streptococcus viruses have dealt with electrolyte requirements and the influence of culture medium on the efficiency of plating (2,3,5,9,19,20). Other ITechnical paper no. 3472, Oregon Agricultural Experiment Station. 72 studies dealing with bacteriophage propagation have not dealt specifically with the development of high titers and have not been concerned with extended survival times of the viruses (10,11,13,18,22). In the present study, two procedures are described for routinely obtaining bacteriophage titers in excess of 1010 plaque-forming units (PFU)/ml. A simple storage procedure also is described which has proven suitable for maintaining these high titers during a 2-year study period. MATERIALS Propagation of phages in milk. Host cultures selected for these experiments were EB4, EB7, EB9, ML1, E8, C2, DRC1, DRC3, C1, C3, Cli, and C13. For each host, eight tubes of litmus milk (10 ml each) were inoculated with 0.1 ml taken from a 24-h litmus milk culture and then incubated at 30 C. Duplicate tubes were infected with 105 to 106 phage particles, at each of four incubation times: 0.5 h, 2 h, 4 h, and 6 h. The infected cultures were incubated overnight at 30 C, and the appearance of each tube was recorded. Sterile lactic acid was added to a final concentration of 1.0% (vol/vol) to aid in whey separation. The contents were centrifuged (15 min at 10,000 x g), filtered through a membrane nitrocellulose filter (Millipore Corp.), 0.45 Mm, to remove any bacterial cells or other debris, and titered for plaque-forming units per milliliter. Influence of buffer on phage growth in lactic broth. Experiments were initiated to study the influence of tris(hydroxymethyl)aminomethane (Tris)-buffered lactic broth on phage propagation. Control experiments indicated that 0.1 M Tris (pH 7.1) did not affect the growth of the host and was effective in maintaining the pH above 6.5 through 3.5 h of growth. A comparative study was made of five phage-host systems propagated in Tris-neutralized or unneutralized lactic broth. Duplicate flasks of each host were infected on the shaker at 34 C with homologous phage; one flask contained Tris buffer at a concentration of 0.1 M, pH 7.1, whereas the second flask of each host remained unbuffered. A 10-ml portion of an overnight culture containing 109 colony-forming units/ml was inoculated into 50 ml of the broth. Flasks were inoculated with homologous phage at zero time. All flasks were titered after 6 h. Effect of polyethylene glycol (PEG) on concentration of phage lysates. Phage lysates were prepared on the shaker in lactic broth made 0.1 M with '+" indicates intensity of reaction; "-" indicates no reaction. Tris-hydrochloride buffer as described in the preceding section. Concentration of the lysates was achieved in an aqueous PEG-NaCI two-phase system. With one exception, the protocol of Yamamoto et al. (23) was followed. The influence of NaCl concentration on phage recovery was studied in the early stages. NaCl was added to some of the lysates in 1 M concentration and to the others in 0.5 M concentration. The 1 M concentration proved more effective. PEG (Carbowax 4000) was added to the supernatant at 10% (wt/vol) immediately after centrifuging in the presence of NaCl. The flasks were stored at 4 C overnight, centrifuged for 20 min at 10,000 x g and decanted. The pellet was suspended in 1/33 of the original volume of lactic broth (1.5 ml), and the phage suspension was titered. Storage by freezing in glycerol. Litmus milk whey lysates containing 15% (vol/vol) added glycerol were frozen at -22 C. They were thawed and refrozen as needed. The lysates were retitered periodically. RESULTS AND DISCUSSION Bacteriophage titers in litmus milk. Preliminary experiments demonstrated that the 'The final plaque-forming units per milliliter and pH were recorded after 6 h of incubation. ' 0.1 M Tris; initial pH with Tris, 7.1; without Tris, 6.6. lactic bacteriophage were quite unstable when stored as low-titer lysates (less than 10' PFU/ ml). For example, when such lysates were maintained in natural or neutralized (0.1 M Tris, pH 7) whey, 50% of the bacteriophage strains could not be recovered after only 7 days at 2 C. For this reason it was necessary to develop a convenient system to achieve and maintain lysates with high titers. Staggered infection in litmus milk was used to explore conditions for obtaining maximum titers, and it was found (Table 1) that the time of bacteriophage addition was critical. By using a 1% (vol/vol) host inoculum and 30 C incubation, highest titers were achieved, with only three exceptions (eb4, c2, and c13), by infecting the culture at 4 h. When infection was delayed until 6 h, there was a dramatic drop in the final titer attained for most bacteriophages examined. At optimum infection times, the final titers were generally 2 to 3 logs higher than that previously reported for lactic bacteriophages propagated in milk (8,11,17,18,19). After 18 h of incubation, many litmus milk cultures containing the highest bacteriophage titers were incompletely reduced, with little or no acid and with slight or no coagulation. On the other hand, host cultures infected at 6 h (low final bacteriophage titers) were completely reduced and firmly coagulated. Thus, the degree of successful bacteriophage propagation could be readily monitored by inspection of the tube contents. Preparation of high titers from lactic broth cultures. Production of milligram quantities of bacteriophage was necessary for the nucleic acid characterization studies now under way in our laboratory. To our knowledge, only the preliminary report of Lowrie deals with the preparation of a large volume of high-titer lactic bacteriophage (16). In that report, dealing only with phage m13, the host was concentrated 10-fold just prior to infection in fresh, doublestrength medium containing 0.005 M calcium borogluconate. Several modifications of this procedure did not yield satisfactory results in our hands with several other phage-host systems. We, therefore, have developed a reliable system for achieving titers of 1010 to 1013 PFU/ml by using buffered lactic broth coupled with the PEG-NaCl concentration technique. The use of 0.1 M Tris was found to maintain the pH near 7.0 without any adverse effects on bacterial growth. Table 2 compares the results of phage infection of homologous hosts in unbuffered or Tris-buffered lactic broth. In each instance, a lower final titer in the absence of added buffer was observed. More significant was the much higher final titers found with c2, clO(1), and eb7 in Tris-buffered as compared with unneutralized lactic broth. These fast acid-producers apparently overwhelm the phage particles with acid and thereby prevent any significant increase in titers. There exists a serious problem then, when a low-titer phage preparation is used for inoculum. In contrast, infection of even fast acid-producers with a few milliliters from a low phage titer lysate in 0.1 M Tris-buffered lactic broth allows maintenance of the pH above 6.0 through several cycles of infection. Thus, final titers greater than 1010 to 1011 PFU/ml are obtained. The effect of pH on production of phage particles has been documented (8,14,19). The current study is in agreement with earlier observations but offers a simple, routine procedure for overcoming the pH problem for preparation of large volumes of lactic bacteriophages in high titer. Concentration of phage lysates. Yamamoto et al. (23) recently reported that PEG-NaCl solutions would allow rapid bacteriophage sedimentation by low-speed centrifugation and that large scale virus purification could be obtained with its application. As shown in Table 3, the average lysate was concentrated 10-fold by using 0.5 M NaCl. With 1 M NaCl, the average concentration was 10-fold, but occasionally reached as high as 100-fold. When using 1 M NaCl, more than 99.9% of the phage were removed from the lysate. Final titers of 1 to 30 x 1010 PFU/ml were routinely achieved. One point of interest was that the lysates suffered up to a 100-fold decrease in titer after standing overnight in 1 M NaCl, before centrifugation or addition of PEG. This indicated that the procedure should not be interrupted before the PEG is added to the salt solution. The use of PEG otherwise proved to be a fast, economical, and efficient technique to obtain large amounts of high-titer lactic phage lysate. In fact, 1 liter of lysate, prepared and concentrated as described above, yielded 2 to 4 mg of DNA. Without PEG, the volume of lysate required to obtain this same yield would be 10 to 50 liters. Preparation of broth lysates in the absence of Tris buffer, particularly from fast-acid producers, would be impractical, requiring from 100 to 10,000 liters of lysate to obtain milligram yields of phage. Storage of lysates in 15% glycerol. Freezing in 15% glycerol was investigated as a means of storage for bacteriophage stocks in whey lysates. This method had been examined by Henning (11) who suggested that phages stored at -20 C in sterile 10% nonfat milk with 15% added glycerol could be recovered after freezing and thawing. Unfortunately, no data were provided to substantiate the usefulness of this menstrum. We have found that, although the percentage of survival varied with each phage, storage of viruses in a 15% glycerol-whey mixture was successful in maintaining stock phage preparations for 2 years or more, even with repeated freezing and thawing (Table 4). Lowas well as high-titer phage suspensions were stored in this manner, and the results indicate that phage lysates may be stored indefinitely in this manner. The procedures described in this study also have been applied to an additional 18 bacteriophages isolated from commercial cheddar cheese whey samples. The results obtained with these phages substantiated the results obtained with the laboratory phage strains reported in this paper.

v3-fos

2020-12-10T09:04:22.987Z

1974-12-01T00:00:00.000Z

237234304

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Nutritional Features of the Intestinal Anaerobe Ruminococcus bromii Of six strains of Ruminococcus bromii studied, five grew in a minimal chemically defined medium containing minerals, NH4+ as nitrogen source, sulfide or sulfate as sulfur source, fructose as energy and carbon source, isobutyrate or 2-methylbutyrate and carbonic acid-bicarbonate as additional carbon sources, and the vitamins biotin, riboflavin, pyridoxine, vitamin B12 (replaced by L-methionine), pantethine, and tetrahydrofolate. The strains also could utilize cysteine or thiosulfate but not methionine; and strain Z3 failed to use dithiothreitol, thioglycolate, sulfite, or β-mercaptoethanol as sole sources of sulfur. Mixtures of amino acids, peptides (Casitone), urea, nitrate, asparagine, or glutamine failed to replace NH4+ as N source. Three strains isolated from Americans were identical in nutritional features, whereas one from a Japanese and one from a South African native differed slightly in having requirements for fewer vitamins. One strain from the cecum of a sow grew well in a rumen fluid-supplemented medium but not in the various chemically defined media plus Casitone. The nutritional features suggest that the environment which selects R. bromii contains relatively little amino acid nitrogen and a relatively large amount of NH4+-N and indicate that these bacteria must depend upon other bacteria such as those that produce NH4+ from urea or protein and those that produce branched-chain volatile acids to grow. Americans were identical in nutritional features, whereas one from a Japanese and one from a South African native differed slightly in having requirements for fewer vitamins. One strain from the cecum of a sow grew well in a rumen fluid-supplemented medium but not in the various chemically defined media plus Casitone. The nutritional features suggest that the environment which selects R. bromii contains relatively little amino acid nitrogen and a relatively large amount of NH4+-N and indicate that these bacteria must depend upon other bacteria such as those that produce NH,+ from urea or protein and those that produce branched-chain volatile acids to grow. Detailed information on the nutritional characteristics of representative, important microbial species of a given ecosystem is essential to an adequate qualitative understanding of the ecology, taxonomy, and metabolism of the species and gives important clues to the chemical nature of the environment which has selected the species. Nutritional information may also be valuable in the development of specific selective cultural media for use in isolation, enumeration, and genetic studies of given species. Although a large amount of nutritional information is available concerning anaerobic species of the rumen ecosystem (4,6,16), nutritional studies on the major anaerobic species of the intestinal tract has only just begun (12,17). In fact, the species of anaerobes of greatest importance in ecosystems, such as the large bowel, are only beginning to be understood (14). Ruminococcus bromii is of particular ecological interest, because it is among the most numerous species in human and swine feces, yet it was not detected until workers initiated studies using special anaerobic techniques and media developed originally for study of rumen anaerobes (13, 14; see also group 10 of Eller et al. [9] and Jennifer Gossling, Ph.D. thesis, West Virginia Univ. , Morgantown, 1973). This organism was probably not found by other workeis because of failure to use adequate anaerobic techniques or to include an energy source used by R. bromii in the isolation medium. It does not utilize amino acids, and usually not glucose, as an energy source but ferments starch and maltose and, usually, fructose. It was also considered possible that it required growth factors present in rumen fluid or fecal extract but of limited occurrence in some other more commonly used crude nutrient sources (13). In the present study, we describe chemically defined and minimal media and indicate some nitrogen and sulfur sources utilized for growth of R. bromii. MATERIALS The anaerobic culture methods uc described by Hungate (11) with modific Inocula for the experimental media as follows. A stab culture, grown ox glucose-cellobiose-starch agar slants (( the refrigerator for 1 day to 2 we inoculated into fresh slants. After incubation at 37 C, the fresh culture i into tubes containing 5 ml of the medium shown in Table 1 suppleme (vol/vol) clarified rumen fluid (8), whir contain other essential factors for grov 24 h of growth, one 4-mm platinum loo] (about 0.01 ml) was inoculated into (13 by 100 mm) containing 5 ml o medium. In an experiment done to df sources, media were used without an; agent such as cysteine or sulfide, inocu per tube so that reducing materials ge inoculum culture reduced the medii transfers of the cultures were made in tal media to dilute out possible sulfur over with the inoculum. Growth was estimated as optical de] with a Bausch and Lomb spectronic-Each value given is a mean for dup medium. Culture purity was checked wet-mount observations with the pha., croscope. The medium listed in Table 1 we experimental media with variations as media and all solutions were prepare( described (3,5,6). Heat labile compot urea, pantethine, and tetrahydrofolic a were prepared n rumen fluid-6) and stored in ?eks, was stab 18 to 24 h of P sterilized, aseptically tubed with CO2 gas phase in 5-ml amounts, and stored in the refrigerator. They were added to previously autoclaved media before it was tubed. was transterred Five of the six strains studied grew well in a basal defined chemically defined medium containing frucnted with 40% tose, minerals, CO2-bicarbonate, cysteine, biwth Aftero18 to otin, riboflavin, pyridoxine, vitamin B12, tetpofthis culture rahydrofolate, pantethine, isobutyric acid, and duplicate tubes methionine ( Table 2). Strains Z3, 6833, and of experimental S6B47 were identical in all growth requirements etermine sulfur studied and all of the B-vitamins or related added reducing factors indicated above were essential. Other lum was 0.1 ml experiments showed that the vitamin B12 renerated by the quirement of these three strains is replaced by um, and serial methionine, as in Bacteroides fragilis (17) and sources carried many other bacteria (12), and that the tetrahydrofolate and pantethine requirements were not nsity at 600 nm replaced by folate and pantothenate, respec-20 calorimeter. tively. Strain J3-2A differed from these three 'licate tubes of strains in that pantethine (or pantothenic acid) periodically by and pyridoxine were not required and the vitase-contrast mimin B12 requirement was not replaced by methionine. Strain A2-6 differed in that neither indicased Trall pantethine (or pantothenic acid), riboflavin, d as previously nor vitamin B12 was required. Strain 0851 grew ands, including well in the inoculum medium which contained cid, were filter-rumen fluid but failed to grow in any of the chemically defined media (Table 2), or in media medium for containing constituents shown in Table 1 Fig. 1) showed that approxi-2.0 (vol/vol) mately 0.05 mM isobutyrate was required for 5.0 (volvol) optimal growth yield and that DL-2-methylbutyrate replaced the requirement for isobutyrate. Bg of KH2PO.; 18 These results also suggest that isovalerate can IgCl2.-6H20; 0.2 g be used in place of the other acids; however, a 10. two-or threefold larger amount was required. nil of acetic acid, Studies with Ruminococcus albus (1) and Baceric acid, DL-2-teroides succinogenes (20) showed that these bacteria have a branched-chain fatty acid reil: 20 mg each of quirement satisfied by either 2-methylbutyrate lride;t1 mg of or isobutyrate, and that activity found in comin and folic acid, mercial isovalerate samples is often due to contamination with D-2-methylbutyrate. VOL. 28, 1974 Whether the present sample of isovalerate had similar contamination was not determined. Results in Fig. 2 and Table 3 show that strain Z3 grew well with NH41 as the nitrogen source and that free amino acids or peptides were not effectively utilized. The growth obtained when-Casitone or Casamino Acids were added to an otherwise low-nitrogen basal medium (Table 3) was undoubtedly due to NH,+ contamination. Based on the growth response of R. bromii on these products compared to limiting levels of ammonia-N (Fig. 2) would be equal to only about 3% or less of the total N present. Other results indicate that the other strains capable of growth in defined media have nitrogen requirements identical to those indicated above for strain Z3. In further experiments, all strains failed to grow in a medium containing a mixture of free L-amino acids (17) in similar proportions to those present in casein and added to give a final concentration of 41 mM amino acid-N, and growth yields in this medium with NH,+ added were essentially identical with or without the amino acid mixture. In an experiment involving strain Z3 only, neither urea, NO,-, NO2-, asparagine, nor glutamine-N were utilized as sole source of nitrogen in place of NH,+. Results in Table 4 show that all of the strains capable of growth in defined media have similar sulfur requirements. They utilized sulfide, sul-fate, thiosulfate, and cysteine as sole sulfur sources. Methionine, as indicated in the studies in which it replaced vitamin Bu,, was utilized but did not serve as sole sulfur source. In another experiment with the same basal medium except that 1 mM dithiothreitol was added as a reducing agent, the above results with strain Z3 were confirmed, and it was further shown that sulfite, thioglycolate, #-mercaptoethanol, and dithiothreitol were not utilized as sole sources of sulfur. The inability of the organism to utilize sulfite seems peculiar in that sulfate is used. One can speculate that a transport system for sulfite is absent in R. bromii. DISCUSSION The results demonstrate that many strains of R. bromii can be grown in a relatively simple minimal chemically defined medium containing minerals, NH,+ as N source, sulfide or sulfate as sulfur source, fructose as energy and carbon source, isobutyrate or 2-methylbutyrate and carbonic acid-bicarbonate as additional carbon sources, and the vitamins biotin, riboflavin, pyridoxine, vitamin B1,, pantethine, and tetrahydrofolate. The strains were selected for study on the basis of diversity of isolation source, and it is of interest that the three strains which were identical in nutritional features were all isolated from Americans (Z3 from a man in Ill., and 6833 and S6B47 from women in Va.). Strain J3-2A, isolated from a Japanese man, and A2-6, from a South African native in the bush, were somewhat different in their patterns of vitamin requirements. The one strain from a non-human source, i.e., strain 0851 from the cecum of a hog, Table 1 plus 0.5 #g of pantethine per ml and 0.09 jig of tetrahydrofolate per ml with changes in (NH4),SO4 and Casitone as indicated above. The 17 mM Casitone-N or Casamino Acids-N was calculated on the assumption that these products contain about 12% nitrogen, i.e., 0.2% was added to the media. ' Values expressed as optical density. Numbers in parentheses indicate hours required to reach maximal growth. failed to grow in the media not containing rumen fluid. Further studies to establish the nutrient requirements of this strain and others from non-human and human sources should be of interest. The nutritional features of this bacterium, which is one of the most numerous intestinal organisms, are surprisingly similar to those of rumen organisms such as R. albus, an important cellulolytic bacterium. The latter organism usually requires 2-methylbutyrate or isobutyrate (1,4). NH4+ is essential as the main nitrogen source, and nitrogen from materials such as amino acids, peptides, nitrate, and urea are not effectively used (4,5). R. albus also usually requires biotin and pyridoxine, but differs from R. bromii in requiring p-aminobenzoic acid but usually not tetrahydrofolate (or folate) or pantethine (or pantothenic acid) (4). R. albus, of course, requires different energy sources such as cellulose or cellobiose. Another anaerobe which is perhaps of intestinal origin, Fusobacterium symbiosum, also requires pantethine (12,15). The fact that R. bromii very effectively utilizes NH,+ nitrogen but not nitrogen of amino acids or peptides suggests that NH,+ is the main nitrogen source available for its growth in the intestinal or cecal environment, i.e., there is probably little survival value in the ability to efficiently utilize amino acids. It is of interest that many rumen bacteria (6,7) also lack the ability to effectively use exogenous amino acid nitrogen. R. bromii is probably dependent upon other bacteria in the tract which produce NH,+ from proteinaceous materials, and upon organisms such as the urease-forming Peptostreptococcus productus (18) which produce NH,+ from urea (19). It probably also depends upon organisms such as B. fragilis, which produce the required branched-chain volatile fatty acids from branched-chain amino acids present in proteins. This is similar to the interaction in the rumen in which organisms such as Bacteroides ruminicola produce the branched-chain volatile acids required by organisms such as R. albus and B. succinogenes (2). The ability of R. bromii to utilize sulfate as sulfur source is of interest. This is done via an assimilatory rather than a dissimilatory sulfate reducing process, as no sulfide can be detected in cultures grown in media containing sulfate (9,13). There is little documentation of assimilatory types of sulfate reducing bacteria among major, strictly anaerobic saccharoclastic bacteria of the gastrointestinal tract. Lachnospira multiparus utilizes sulfate (10) but other

v3-fos

2020-12-10T09:04:12.932Z

1974-02-01T00:00:00.000Z

237229724

s2

Comparison of Fluorescent-Antibody Methods and Enrichment Serology for the Detection of Salmonella Four rapid methods for detection of Salmonella, (i) the conventional fluorescent-antibody (FA) technique, (ii) a rapid direct FA technique, (iii) microcolony FA, and (iv) enrichment serology (ES), were compared with conventional cultural procedures. A total of 347 subsamples representing 16 different food prototypes, alleged to be naturally contaminated with Salmonella, were analyzed. From these samples, 52 were found to contain Salmonella by cultural methods. Conventional FA identified all 52 culturally positive samples, ES identified 51, microcolony FA identified 48, and the rapid FA method identified 34. The number of false-positive samples for each procedure was: ES-selenite, 7; tetrathionate, 8; rapid FA, 26; microcolony FA, 33; conventional FA-selenite, 27; tetrathionate, 26. Tetrathionate enrichment was found to be superior to selenite for Salmonella recovery from most foods, but the concurrent use of both media allowed maximum recovery. The demand for routine analysis for the detection of Salmonella in food and feeds has increased significantly in the last few years. As a result of this increased testing, research has been directed toward the development of faster and/or more sensitive methods to detect these organisms. The use of the fluorescent-antibody technique (FA) is not new, and recently two modified FA procedures for the detection of Salmonella have been described (5,12). In addition, an enrichment serology (ES) method (1,2,10) has been evaluated and reported for use as a quality control measure in bacteriological laboratories. The primary objective of this study was to compare five different methods for the detection of Salmonella in food and feeds. These methods were: (i) the conventional FA technique; (ii) a rapid direct technique described by Insalata et al. (5); (iii) the microcolony technique as described by Thomason (12); (iv) the ES technique as proposed by Sperber and Deibel (10); and (v) the standard cultural methods (13), with minor modifications. The procedure referred to as "conventional FA technique" is a modification of direct FA staining as described by numerous investigators (3,6,9). The experimental design used allowed a comparison of the time for pre-enrichment (7 versus 24 h) and an evaluation of the efficiency of tetrathionate and Selenite-F selective enrichments. MATERIALS AND METHODS Food and feed samples suspected of being naturally contaminated with Salmonella were obtained from various sources. The products, number of lots, and subsamples analyzed are presented in Table 1. Two sources of commercially available FA antisera were used throughout this work: FA Salmonella poly antiserum (11) (Difco, Detroit, Mich.) diluted 1:4 with sterile saline, and Salmonella Fluoro-Kit (4) (Clinical Sciences Inc., Whippany, N.J.) prepared as directed by the manufacturer. No differences between antisera were noted. Pre-enrichment. Samples (25 g) of each food or feed prototype were pre-enriched in 225 ml of FAS broth (Difco) which was tempered at 35 ± 2 C. Milk products were pre-enriched in brilliant green water as recommended in the Bacteriological Analytical Manual (13). Incubation was at 35 ± 2 C for 24 h, with the exception of the rapid FA technique for which the incubation time was 7 h (5). Incubation was without agitation. Selective enrichment. After incubation, the preenrichments were shaken and allowed to settle for about 5 min. In the rapid FA technique, 50 ml was transferred to 450 ml of prewarmed (35 ± 2 C) Selenite-F broth (BBL) and incubated for 16 to 18 h at 35 ± 2 C without agitation. For all other tests, 2-ml portions of the pre-enrichment broth were transferred to 18 ml of Selenite-F broth and 18 ml of tetrathionate broth, respectively, and incubated at the same temperature for 24 h. Elective enrichment. Selenite-F broth was agitated and allowed to settle for 5 min for the rapid FA technique. Two milliliters was then withdrawn from 324 Cereal-spice mix 1 20 a Number of samples confirmed positive. the top third of the culture, transferred to 18 ml of tempered FAS broth, and incubated for 5 h at 35 4 2 C. For conventional FA analysis, 1 drop from Selenite-F and 1 drop from tetrathionate broth were transferred separately into 3 ml of Trypticase soytryptose broth (TST) (7) and were incubated at 35 2 C for 3 h or until visible growth was obtained. This elective enrichment medium for conventional FA was suggested by Wallace H. Andrews, Jr., of the Food and Drug Administration. The use of an elective enrichment in FA analysis resulted in cleaner slides with less background debris. For the enrichment serology technique, 1 drop from each selective enrichment culture was added to M broth and incubated for 6 h at 35 2 C in a water bath. Serological analysis was performed as recommended by Sperber and Deibel (10). FA staining procedure. A loopful (2 mm) of the appropriate elective enrichment broth was placed onto the Fluoro-Kit slides, and they were stained by using the materials and methods specified by the supplier. If it is desired to observe nonfluorescent cells, FA Rhodamine counterstain (Difco) may be applied for 1 min after FA staining and then slides are rinsed for 30 s with distilled water. All nonfluorescent cells would be stained red. For the microcolony technique, a 2-mm loopful from tetrathionate enrichments was placed onto brilliant green agar plates in such a manner as to coincide with the prepared areas on the Fluoro-Kit slides. The plates were incubated for 3 h at 35 i 2 C. After this time, impression smears were made by placing the slides on the agar plates and exerting slight pressure. Slides were removed, allowed to airdry, and were then stained by the recommended method. Microscopy examination. Slides were examined on a Leitz Ortholux microscope equipped with incident-light fluorescence using exciting blue filters KP490 (FITC) and dichroic beam splitters with a K495 built-in suppression filter and 510 suppression filter slide. The light source was an Osram HBO-200 mercury arc burner equipped with a transmission heat filter (2 mm KGl) and a red suppression filter (4 mm BG38). Smears were examined by using a dry x40 objective and x 16 oculars. A x54 oil fluorite objective was used for more detailed examination of smears to confirm the presence of attached flagella. Approximately 100 microscopic fields were scanned on each slide. One or more strongly fluorescing rods with discernable lumen in 10 or more fields was considered as positive. Cultural methods. Selective enrichments of Selenite-F and tetrathionate broths were streaked on XLD agar (Difco) and hectoen enteric (HE; Difco) agar (8). XLD and HE agars were incubated at 35 4 2 C for 24 h. In our laboratory, we found XLD and HE to be as good or better than Salmonella-Shigella agar and brilliant green agar for the isolation of Salmonella. Typical colonies were picked to triple sugar iron agar and lysine iron agar, and cultures exhibiting a presumptive positive Salmonella characteristic were confirmed serologically. Subcultures in TST broth from Selenite-F and tetrathionate were incubated at 35 2 C for 7 h and then streaked on XLD and HE agar. The experimental procedures are illustrated schematically in Fig. 1. RESULTS Out of 347 subsamples, 52 were positive by the cultural method employed in this study. Five of the isolates were not recovered from selenite enrichment but were recovered from tetrathionate enrichment. One of these five cultures was isolated from tetrathionate only after the additional elective enrichment in TST broth. This isolate would have been missed by routine cultural methods. Another isolate did not grow in tetrathionate broth but was recovered from selenite broth ( Table 1). The conventional FA method correctly identified 48 positive samples from selenite enrichment and 52 from tetrathionate enrichments. The rapid FA method correctly identified 34 positive samples. Microcolony FA detected 48 of the 52 positive samples. The ES method gave 43 and 50 positive results from selenite and tetrathionate, respectively. Since the ES method calls for analysis of both selective enrichments, the method detected a total of 51 positives (Table 1). In the following discussion, a false-negative result is defined as a culturally positive sample which was negative for Salmonella by either FA or ES methods. A false-positive result is defined as a culturally negative sample which exhibited a presumptive positive test for Salmonella by either FA or ES methods. The rapid FA procedure gave 18 false-negative results compared to 4 false negatives for conventional FA from selenite enrichment. No false-negative results were obtained with the conventional FA technique using tetrathionate enrichment. Microcolony FA resulted in four false negatives. The enrichment serology method showed nine and two false negatives from selenite and tetrathionate, respectively. Since the method calls for analysis of both enrichments, the use of the ES technique resulted in only one false negative ( Table 2). False-positive results were found with all methods. However, the number was quite different for the various procedures. The lowest was ES, with seven and eight from selenite and tetrathionate, respectively. Conventional FA showed 27 and 26 from selenite and tetrathionate. The microcolony method resulted in 33 false positives, and rapid FA had 26 (Table 2). DISCUSSION Any screening procedure for Salmonella must be as sensitive as standard cultural methods. This means the procedure should not result in any false negatives. However, as a practical quality control method, it should also not produce excessive false-positive results, since these require lengthy and costly cultural confirmation. The rapid FA method resulted in 18 false negatives. This was probably due to the shortened pre-enrichment phase. When the same samples were analyzed by conventional FA using 24-h pre-enrichment followed by selenite selective enrichment, only four false negatives were obtained. Since the rapid FA technique relies on selenite enrichment, 5 of the 18 falsenegative results can be explained by the failure of the Salmonella in those samples to grow in selenite. The conventional FA procedure using tetrathionate selective enrichment resulted in no false negatives. In one sample Salmonella was not recovered from tetrathionate culturally, yet the conventional FA produced a positive test. This positive result was confirmed by the 326 irs. use of the elective TST enrichment, which is not part of the AOAC method. The number of Salmonella cells in the tetrathionate enrichment was too low to be detected on streak plates, but cells were found in FA smears. The false-positive rate found with conventional FA using the TST elective enrichment is comparable to that reported without the use of elective enrichment (5). In the microcolony technique, 24-h preenrichment was combined with tetrathionate enrichment, resulting in four false negatives. Some of these false negatives may have resulted from excessive growth on the brilliant green agar elective enrichment, creating a "quenching" of fluorescence on the slides. Reducing the incubation time of the brilliant green agar plates may overcome this problem. The microcolony technique did not offer any time saving over other methods. Microscopy examination of all FA smears was facilitated by the use of incident light illumination. Fluorescence was extremely bright and easily seen under x640 magnification. Since no dark-field condenser was needed, adjustment or oiling of the substage condenser was not necessary, thereby saving time and avoiding the possibility of inaccurate adjustment which would reduce brightness of fluorescence. The ES method using both enrichments gave only one false-negative result. The amount of growth in the elective M-broth enrichment was very scant in this instance and may have been the cause of this single failure. The need for a longer incubation time for M broth has been recently demonstrated (1). In the opinion of the authors the ES technique, using 6-h elective enrichment, is the preferred Salmonella screening method. Incubation of the elective enrichment must be extended in cases where culture turbidity is insufficient for serological testing. The ES method is simpler to perform, gives fewer false-positive results, requires less costly equipment, and requires significantly less technical expertise. The ES results found in this study are similar to those seen by Sperber and Deibel (10) and more recently by Boothroyd and Baird-Parker (1). Conventional FA would be the method of second choice. It has the advantage of detecting low numbers of Salmonella cells from elective enrichment broth. This sensitivity was demonstrated by the absence of false negatives when tetrathionate enrichment was used. However, due to numerous false positives, additional cultural confirmation would be required. The rapid FA procedure was found to be inadequate because incubation of the preenrichment broth for 6 h was insufficient. Fourteen additional Salmonella-positive samples were found when pre-enrichment incubation was extended to 24 h. If only one selective enrichment broth is to be used, then tetrathionate would be the broth of choice. However, in these studies limiting the enrichment to just tetrathionate would have missed two positive samples that were isolated from selenite broth. Studies are currently under way to evaluate the use of antibiotics and elevated temperatures for enhancement of Salmonella isolation.

v3-fos

2020-12-10T09:04:12.370Z

1974-04-01T00:00:00.000Z

237233304

s2

Some Factors Affecting the Viability of Dried Bacteria During Storage In Vacuo The effects of various substances on the viability of freeze-dried cells of Pseudomonas fluorescens and Salmonella newport were studied during storage in vacuo for 5 years. Mixtures of two organisms were dried together and studied in two factorial experiments. The first was a complete factorial using six factors and two levels; the second was a fractional replicate with four factors at two levels and three others at four concentrations. A study of ribose binding by cells of S. newport was made by using [U-14C]ribose. Substantial improvements in viability were obtained by drying in the presence of sucrose, glutamate, and semicarbazide. Low concentrations of ribose increased the death rate during drying, but these adverse effects were prevented by equimolar amounts of semicarbazide. Ribose binding increased with storage time and, although its incorporation changed, most of the increase in total ribose occurred after the main decrease in viability. Storage temperature caused larger changes in viability than did level changes of residual water in the cells. Although the results are complicated by a large number of interactions, they confirm and extend the hypothesis that reactions between carbonyl compounds and cellular components are a major cause of the mortality occurring during storage of dried microorganisms. A particular mixture of sucrose, glutamate, and semicarbazide is proposed as a means of reducing death when prolonged storage in the dry state is desired. The effects of various substances on the viability of freeze-dried cells of Pseudomonas fluorescens and Salmonella newport were studied during storage in vacuo for 5 years. Mixtures of two organisms were dried together and studied in two factorial experiments. The first was a complete factorial using six factors and two levels; the second was a fractional replicate with four factors at two levels and three others at four concentrations. A study of ribose binding by cells of S. newport was made by using [U-14C Iribose. Substantial improvements in viability were obtained by drying in the presence of sucrose, glutamate, and semicarbazide. Low concentrations of ribose increased the death rate during drying, but these adverse effects were prevented by equimolar amounts of semicarbazide. Ribose binding increased with storage time and, although its incorporation changed, most of the increase in total ribose occurred after the main decrease in viability. Storage temperature caused larger changes in viability than did level changes of residual water in the cells. Although the results are complicated by a large number of interactions, they confirm and extend the hypothesis that reactions between carbonyl compounds and cellular components are a major cause of the mortality occurring during storage of dried microorganisms. A particular mixture of sucrose, glutamate, and semicarbazide is proposed as a means of reducing death when prolonged storage in the dry state is desired. In a recent paper, Marshall et al. (2) reported on the effects of various gases on the preservation of dried bacteria during storage at controlled levels of water activity (a,). The present paper reports the principal results of similar experiments studying the effects of various substances on the death rates during storage under controlled conditions for periods up to 5 years. The substances selected previously were found by Scott (5) to affect the death rate during storage. He first hypothesized that a major cause of death in dried organisms was due to reactions between carbonyl compounds and amino side chains of cellular components including proteins. A preliminary account of some results has already been reported (4), and a fuller account giving details of the changes during drying as well as storage, the statistical procedures, tables showing the nature of many of the interactions, and appendices giving all estimates of viable cells has been documented elsewhere (3). The results were obtained from two factorial experiments. The first was a complete factorial for six factors at each of two levels. These included sucrose, glutamate, and semicarbazide as factors previously found to reduce mortality, and ribose as a substance that increased deaths during storage; storage was in vacuo at two temperatures and two levels of a,. The second experiment was a fraction of a 21" factorial in which the factors sucrose, glutamate, and semicarbazide were studied at four levels and Llysine, DL-a-alanine, aw, and ribose at two levels. MATERIALS AND METHODS Organisms. The organisms used in the two experiments were Pseudomonas fluorescens and Salmonella newport. Factorial designs. The first experiment was a single replicate of a 26 factorial with the following factors and levels: Preparation for drying and storage. The two organisms were dried together in the particular solute mixture and stored as described by Marshall et al. (2). Saturated lithium chloride with crystals was used instead of a sulfuric acid solution in the second experiment to adjust the aw to 0.11. All the treatments were stored in vacuo in the dark in insulated cabinets at the particular temperature. Viable counts. Viable counts were made before freeze drying, after drying, and after storage for 3, 9, 27, 81, and 243 weeks (256 in the second experiment) according to the method previously described (2). The viable numbers per milliliter were expressed as twofigure logarithms derived from the mean counts of duplicate plates. Determination of ribose uptake by cells during storage. [U-'4CIribose was used to study uptake of ribose by dried cells of S. newport during storage. The ratio by weight of ribose to [U-"4Clribose was 100 to 1 at all levels of ribose. Portions of 1 ml of the cell/solute mixtures were dried and stored under controlled conditions (2). In making determinations, the driedcell pellet was rehydrated with 10 ml of saline, and 0.1-ml portions of the suspension were used for the viable counts. After washing twice and suspending to 10 ml with saline, the`C count was determined on a 1.0-ml sample of the washed cells and an estimate for the total "4C was calculated. The washed cells from the remaining 9.0-ml portion were ruptured using 20-g ballotini beads and shaking for 0.5 h at 940 reciprocations/min. The beads were removed by filtration and the cell walls by centrifugation. The protein fraction was precipitated with 5% trichloroacetic acid and centrifuged out. After removal of most of the water from the nonprotein fraction, the determinations of l4C in the three fractions were made. All data have been corrected to give "4C counts per minute for total cells per ampoule. RESULTS AND DISCUSSION First experiment. All the average main effects were very significant at the end of the storage for 243 weeks; this is shown by the mean log counts per milliliter in Table 1. The general effect was that sucrose, glutamate, and semicarbazide were protective; ribose was damaging; storage at 10 C was better than at 30 C; and storage in the dry state better than at 0.20 a,. These effects were conditioned by many interactions, the natures of which are shown by the means in Table 2. There were more interactions for P. fluorescens than for S. newport, which maintained the higher average viability. Second experiment. The 1/16th fraction gave only a small number, 64, of the full set, 1024, of treatment combinations so that there was no clear estimate of error, and not all first-order interactions were free from confounding. Hence, only the average main effects are given; they are shown by the treatment means in Table 3. It appears that the addition of sucrose, glutamate, lysine, alanine, and semicarbazide was beneficial, ribose was damaging, and the levels of a, used made little difference. The highest levels of sucrose and glutamate did not give any additional benefit. There was evidence of interaction between certain factors, just as in the first experiment, so these statements of effects require some qualification (3). P. fluorescens again suffered the greater mortality. Figure 1 plots the mean log counts for the 16 combinations of concentrations of glutamate and semicarbazide for P. fluorescens for two separate levels of ribose and four periods of storage. From the top pair of diagrams in Fig. 1 it is clear that immediately after drying there is little effect of either glutamate or semicarba- zide. The second pair of diagrams shows a greatly changed situation after storage for 27 weeks. After this period either substance reduced mortality when no ribose was added, but in the presence of 0.02 M ribose, glutamate was less effective unless some semicarbazide was also included. With more prolonged storage, glutamate continued to be effective when no ribose was added, but in the presence of added ribose, glutamate declined in efficiency and semicarbazide became beneficial at all levels of glutamate. An adequate interpretation of these complex interactions must await much more detailed information about the chemical changes occurring in cells dried in the presence of various substances. Results with S. newport showed similar trends but lower average mortality. Takano and Terui (6) showed that radioactive ribose was partly bound to cells of S. cereviseae during primary and secondary drying. When S. newport cells were dried in the presence of 0.02 M [U-4C ]ribose and stored for short periods at 0.30 a, at 25 C, there was no detectable binding of ribose during drying, although substantial binding of ribose into the dried cells occurred after storage for only 7 days. The increase of radioactivity approached a maximum after about 9 weeks of storage when 60 to 70% of the bound ribose was found in the intracellular protein fraction, 20% in the nonprotein fraction, and 5% in the cell walls as shown in Fig. 2. It is also evident from Fig. 2 that the initial high rate of binding of radioac-tive ribose occurred when the rate of loss of viability was greatest. Other experiments showed that the addition of glutamate or sucrose (0.25 M) did not depress the rate and extent of ribose uptake even though viability was increased. On the other hand, the addition of semicarbazide prevented binding of ribose under similar conditions of storage. These results suggest some differences in the mechanism by which these three substances protect dried cells during storage. It was also shown that when S. newport was dried in 0.1 M sucrose and 0.1 M ribose and stored in vacuo over P205 for 9 weeks at 25 C, viability was reduced from 10.73 to 5.60 and some 5% of total ribose was bound by the cells. It is evident that these very dry storage conditions did not entirely prevent binding of ribose by the cells. With our present limited understanding of the chemical events leading to death of dried microorganisms it would seem wise to depend on mixtures of two or more protective substances whenever it is desired to prevent death during prolonged storage in the dry state. Several of the most effective mixtures have already been listed by Marshall and Scott (4), who pointed out that these always included at least one amino acid and usually some semicarbazide. A practical recommendation arising from these results is the use of a particular suspending medium when bacterial cultures are being prepared for storage in the dry state. The most useful mixture might contain 0.1 M sucrose, 0. justed to about pH 7. Storage in vacuo at a temperature of 10 C or lower and at levels of residual moisture corresponding to 0.1 a, should also assist when prolonged storage is

v3-fos

2018-04-03T04:11:31.648Z

1974-04-01T00:00:00.000Z

22205851

s2

Form of Selenium in Selenite Enrichment Media for Isolation of Salmonellae Selenite-F and selenite-cystine media, commercially available for the routine isolation of salmonellae, were treated by anion exchange chromatography to separate the selenium from other components of the media. A chemical assay, based on an ascorbic acid reduction, showed that the selenium was all in the form of selenite. NaHSeO2 and an undefined mixture of polypeptones; other substances such as cystine, which apparently enhance the selectivity of selenite (6), are sometimes included. A reaction between selenite and low-molecular-weight thiols is well established (1), and a stoichiometric reaction between selenite and the thiol groups of proteins has also been described (2). Smith (10), on the basis of growth studies with these media, concluded that selenite reacts with and becomes bound to other components of the selenite medium and that the resulting seleniumcontaining substances, rather than selenite itself, are the ones that inhibit the growth of sensitive organisms. A direct analysis to determine the exact nature of the selenium, however, has not been reported. This note will present evidence that the selenium in both Selenite-F and selenite-cystine media remains as selenite. Selenite-F and selenite-cystine media were purchased from BioQuest, Cockeysville, Md. Selenite-F medium was prepared without heating, according to the manufacturer's directions. 7"Se-selenious acid was obtained from Union Carbide Corp., Tuxedo, N.Y., and from Amersham/Searle Corp., Arlington Heights, Ill. To remove small amounts of radioactive contaminants, the 75Se-selenious acid was purified by anion exchange chromatography (9). To determine whether the selenium present in the selenite medium was readily precipitable by ascorbic acid, and to determine the amount of such selenium, a selenium assay adapted from an ascorbic acid reduction method (A. A. Tumanov, N. M. Shakhverdi, and Z. I. Glazunova, Chem. Abstr. 67:3731, 1967) was used. To 2-ml samples that contained from 0 to 200 ,ug of selenium were added 2 ml of 2 N HCl followed by 2 ml of a saturated solution of reduced ascorbic acid. After 5 min, each tube was stirred with a Vortex mixer. Transmission was measured either with a Spectronic-20 at 500 nm or with a Klett-Summerson colorimeter with a blue filter (no. 42). The standard curve with NaHSeO, was linear over a range from 0 to 160 ,ug of Se/ml. The selenium recovered from Selenite-F medium equaled the amount specified by the manufacturer. Unheated selenitecystine broth gave identical results. To determine if selenite becomes associated with components of Selenite-F medium, a mixture of 75Se-selenious acid and medium was examined by gel filtration chromatography on a Sephadex G-10 column. Materials that absorbed at 280 nm (A280) were monitored with a Beckman DB-G spectrophotometer. Radioactivity was monitored with a Packard gamma spectrometer. Figure 1 shows that the peak of radioactivity failed to coincide with any of the A280 peaks. Separation of selenite from A280 materials by anion exchange chromatography is illustrated in Fig. 2. Selenite powder was prepared as described above. Much of the A280 material was VOL. 27, 1974 removed with water; after A280 had reached a minimum, HCl of pH 2.0 was applied, and the remainder of the A28, material was removed. Radioactivity was eluted by HCl of pH 1.5; it emerged at the position of purified 75Se-selenious acid. No radioactivity was detected in any of the other fractions. A small rise in A280 readings consistently preceded the radioactive peak in each experiment. However, no clear-cut peak accompanied the peak of radioactivity (insert, Fig. 2). A ninhydrin reaction carried out on the radioactive fractions was negative. Identical results were obtained with unheated selenite-cystine medium. Storage of both types of medium in the freezer for several months had no effect on the elution pattern. To determine how much selenium was actually present in the radioactive fractions, they were pooled, and samples were analyzed for selenite by the ascorbic acid reduction method. The selenium recovered in the pooled fractions accounted for all of the selenium that had been placed on the ion exchange columns. The ready precipitability by ascorbic acid is one indication that selenium in Selenite-F and selenite-cystine media is still in the form of selenite. Under the acid conditions of this assay, organic trisulfides (R-S-Se-S-R), known to form when sulfhydryl-containing compounds are treated with selenite, would probably be stable (1,2). The absence of radioactive peaks in 815 FIG. 2. Ag-i-Cl chromatography of Selenite-F medium. Two milliliters of a solution containing 183.7 mg of the medium (x4 the normal concentration) and 75Se-selenious acid as marker was applied to a column of Ag-1-x8, 200-400 mesh, Cl-form, 1 by 56 cm. The column was pretreated with 1 N HCI followed by glass-distilled water until free of A28, materials and until a pH of about 4 to 5 was reached. Two-milliliter fractions were collected. association with A28, peaks in the Sephadex G-10 and anion exchange eluates, as well as the quantitative recoveries of radioactive selenium in the radioactive peaks, show a failure of selenite to react with components of the media. The anion exchange chromatographic separation of selenite from A280 materials and the quantitative recovery of total selenium in the resolved radioactive peaks support this conclusion. Any explanation for the unusual tolerance shown by salmonellae, therefore, must be sought, not in some other form of selenium as has been claimed (10), but in the biochemical properties of selenite.

v3-fos

2018-04-03T01:46:04.745Z

1974-01-01T00:00:00.000Z

31044907

s2

Evaluation and modifications of media for enumeration of Clostridium perfringens. The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfitecycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. SPS agar selectively inhibits growth or interferes with the formation of black colonies by the sulfite-reducing Enterobacteriaceae and Achromobacteriaceae; it also inhibits growth of most other facultative anaerobes and of the genera Pseudomonas, Bacillus, and Lactobacillus (1,5). However, low recoveries of C. perfringens in commercial SPS agar have been reported (12; L. F. Harris and J. V. Lawrence, Bacteriol. Proc. 70: 6,1970). Hauschild et al. (7) recovered 12 strains of C. perfringens quantitatively in SPS agar prepared in the laboratory from its ingredients, but in only one out of four commerical lots of SPS. Handford and Cavett (4) and Harmon et al. (5) also obtained low recoveries in laboratory-prepared SPS agar. In this laboratory, we have usually obtained complete recoveries of C. perfringens in SPS agar prepared from its ingredients, but in two preparations the recoveries of some C. perfringens strains were below 1% (D. Dobosch and A. H. W. Hauschild, unpublished data). In one preparation, the cause could be traced to a particular lot of yeast extract. It appears that the selective ingredients of this agar are at a level where a slight adverse change in the medium may result in inhibition of C. perfringens. TSN agar has been used less extensively than SPS agar, but the few reports on the suitability of this medium indicate that it is inhibitory to a number of C. perfringens strains (4, 5; Harris and Lawrence, Bacteriol. Proc. 70: 6,1970). SFP agar appears to allow quantitative recovery of C. perfringens (4)(5)(6)12). Unfortunately, it does not prevent growth of a large number of facultative anaerobes, some of which are sulfite reducing (6,12). Its applicability, therefore, seems to be limited to specimens in which C. perfringens is the predominant microorganism, i.e., foods responsible for C. perfringens enteritis or fecal samples from patients recovering from the disease. The use of neomycin-blood agar commonly used in the United Kingdom (8,14) is similarly limited to investigations of food-poisoning incidents. Another disadvantage of the SFP agar is its relatively elaborate preparation: it requires addition of fresh egg yolk, surface plating, and pouring of cover agar. Harmon et al. (6) modified SFP agar by replacing polymyxin B and kanamycin with 0.04% D-cycloserine. This antibiotic had been shown to selectively inhibit growth of essentially all of the common facultative anaerobes (2). In this modified medium (TSC agar), each of 10 C. perfringens strains tested was enumerated quantitatively by Harmon et al. (6). Presumptive enumeration of C. perfringens is followed by confirmatory tests. The simplest of these involves stab culturing of an adequate number of black colonies into nitrate motility (NM) agar (1). However, the nitrite test as described by Angelotti et al. (1) is unreliable (3,12). Shahidi and Ferguson (12), therefore, intro-78 on March 23, 2020 by guest http://aem.asm.org/ Downloaded from duced egg yolk into their medium and proposed to enumerate only black colonies with an opaque halo around them and to confirm these in lactose motility (LM) agar. Of the clostridial species that produce sulfide as well as lecithinase, only C. perfringens is nonmotile and lactose positive. ln our experience, this method has the following main shortcomings: (i) several C. perfringens species do not produce a discernible halo after 20 to 24 h of growth in SFP and TSC agars; (ii) due to excess gas formation in LM agar, nonmotility of these isolates is difficult to ascertain. This work was initiated to evaluate the suitability of the SFP and TSC agars for enumeration of C. perfringens and to determine the conditions required to obtain consistent results in the nitrite motility test. While this work was in progress, Handford and Cavett (4) published a note on the enumeration of C. perfringens in OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agar. An evaluation of this medium is included in the present paper. MATERIALS AND METHODS Cultures. Seventy-one strains of C. perfringens were examined; 51 of these were isolated from foodpoisoning incidents, 11 from pathological specimens, 7 from soil and normal feces, and 2 were of unknown origin. Strains were supplied by C. R. Amies, Willowdale, Ontario (six strains); R. J. Avery, Hull, Quebec The working cultures were preserved in 15% glycerol (10) at -18 C; they were thawed, inoculated into screw-cap test tubes containing 15 ml of cooked meat medium (Difco), and incubated at 37 C for 20 h. Enumeration procedures. The cultures were diluted in 0.1% peptone (13). When egg yolk-containing media were used, 0.1-ml volumes of diluted culture were spread on the agar surface in standard petri plates. Two plates were used per dilution. When completely dry, the surface was covered with about 10 ml of cover agar. Egg yolk-free media were used in pour plates with 1.0-ml volumes of diluted culture per plate. All plates were incubated anaerobically at 37 C for 20 h. All plating media were prepared from the same agar base consisting of 1.5% tryptose (Difco), 0.5% Soytone (Difco), 0.5% yeast extract, 0.1% ferric ammonium citrate (British Drug Houses), 0.1% sodium metabisulfite (Na2S2O5; British Drug Houses), and 2% agar. The ingredients were dissolved in distilled water to either 92% of the final volume to allow for subsequent addition of egg yolk suspension, or to the final volume. The pH was adjusted to 7.6 before addition of the agar. The agar base was also obtained commercially (SFP agar base, Difco). Antibiotics and egg yolk suspension were added to the autoclaved medium at 50 C. SFP agar. Complete SFP agar was prepared by adding to 920 ml of agar base: the contents of one antimicrobial vial P (30,000 U of polymyxin B [Difco] in 10 ml of distilled water); 4.8 ml of the contents of an antimicrobial vial K (25 mg kanamycin [Difco] in 10 ml of distilled water); and 80 ml of egg yolk suspension containing one egg yolk per 20 ml of 0.85% NaCl. The SFP cover agar had the same composition as the complete SFP agar, except that it contained no egg yolk. Media with D-cycloserine. The second group of plating agars contained varying amounts of D-cycloserine (D-CS; Nutritional Biochemical Corp., Cleveland, Ohio) instead of polymyxin B and kanamycin (6). The medium containing 400 gg of D-CS per ml (0.04%) is identical with the TSC agar of Harmon et al. (6). The antibiotic was added as a 4% filter-sterilized solution in water. The plating procedure was as described for the SFP agar. The third group of plating agars differed from the second in two aspects: no egg yolk was added, and they were used in pour plates only. OPSP agar. Details for the preparation of OPSP agar not contained in the note of Handford and Cavett (4) were obtained by personal communication. The basic ingredients, including ferric ammonium citrate and sodium metabisulfite, were the same as in the SFP and TSC agars. The final concentration of sodium sulfadiazine 272 molecular weight (American Cyanamid Co., Pearl River, N.Y.) was 109 mg/liter, which corresponds to 0.01% sulfadiazine (250 molecular weight) used by Handford and Cavett. Concentrations and origins of the antibiotics were the same as in the work of these authors; the final concentrations of oleandomycin phosphate (Pfizer Co., Montreal) and polymyxin phosphate (aerosporin; Burroughs Wellcome Co., Montreal) were 0.5 mg/liter and 10,000 IU (equivalent to 1.0 mg of polymyxin standard) per liter, respectively. The OPSP agar was used without egg yolk and in pour plates. The results of all enumerations were expressed as percentages of the counts in the corresponding antibiotic-free control medium. Confirmatory tests. Single colonies were stabinoculated into LM agar (12), NM agar (1), and NM agar supplemented with 0.5% each of glycerol and galactose (11) (12) and of Harmon et al. (5,6), but the experiment revealed two considerable shortcomings of both media. (i) Most C. perfringens strains produced large colonies in SFP and TSC agars; counts of over 50 per plate therefore became progressively inaccurate, and 10-fold dilutions were often inadequate. (ii) Of 21 strains, 8 had no discernible opaque halos around the black colonies after the first day of incubation; presumably, such colonies would not be counted in the procedure of Shahidi and Ferguson (12). These drawbacks as well as the lengthy plating procedure are all associated with the dependence of the method on the egg yolk reaction which had been introduced because the nitrite motility test was unreliable. The following experiments were designed to determine the conditions (i) for quantitative enumeration of C. perfringens in a selective medium without egg yolk and (ii) for consistent nitrite reactions in the confirmatory tests. Enumeration in egg yolk-free agar with D-CS. Table 1 shows the recoveries of 71 strains in egg yolk-free agar with different concentrations of D-CS. The recoveries were essentially quantitative at D-CS concentrations of 200 and 400 jig/ml; the lowest count at 400 Ag/ml was 64% of the count in the control medium. Several strains were partially or totally inhibited at D-CS concentrations of 600 and 800 jig/ml, with mean recoveries of 63 and 39%, respectively. About 40% of the strains listed in Table 1 were tested in medium with the commercial agar base; due to supply problems, the remaining strains were tested in medium prepared from the ingredients. This did not appear to affect the results. A detailed table showing the recoveries of each strain is available upon request. At all concentrations of D-CS (Table 1), the strains produced only black colonies, even at the surface. However, a few strains occasionally produced colonies at the surface with a narrow, white halo around the black center. The medium with the highest D-CS concentration (400 jig/ml) that does not significantly Table 2. Of the 142 isolates (duplicate colonies of the 71 strains), only 112 showed a positive nitrite reaction in basic NM medium. Five tubes contained traces of nitrite as evidenced by a faint red color, and 25 tubes contained no detectable nitrite. Most of the negative tubes showed only little growth. All of the isolates grown in supplemented NM medium showed good growth and produced positive nitrite reactions; most of these were very intense, in contrast to the reactions in basic NM medium ( Table 2). Two strains each of C. sporogenes and C. bifermentans were used as negative controls; none of these showed a color reaction. During incubation, the pH of the C. perfringens stab cultures dropped from 7.1 to 6.7-6.9 in NM agar and to 5.6-6.1 in supplemented NM agar. Uninoculated tubes with supplemented NM agar, adjusted to pH 5.5, were therefore incubated as additional controls; they were all negative for nitrite. All NM media were used within 2 weeks after preparation, and all were de-aerated before stabbing. In a separate experiment, we compared the suitability of freshly prepared, supplemented NM medium with the same medium stored for 5 weeks at 4 C. No difference was found. In contrast, basic NM medium deteriorated rapidly during storage: in the fresh medium, 24 out of 28 isolates showed positive nitrite reactions, and 4 gave trace reactions; in the same medium stored for 3 weeks at 4 C, only two isolates produced nitrite; the remaining 26 were negative for nitrite. Comparison of surface-plated egg yolk media with egg yolk-free pour media. We compared the recoveries of 19 C. perfringens Table 1) and in surfaceplated medium with egg yolk and cover agar (6). The SFP medium was included for comparison. In both media containing 400 Ag of D-CS per ml and in SFP agar, the recoveries were essentially quantitative (Table 3). At the higher concentrations of n-CS, the recoveries in medium with egg yolk were considerably lower than in egg yolkfree medium. The differences were likely due to exposure of the C. perfringens cells to high oxygen tension in the surface plating procedure. The results demonstrate that the recoveries of C. perfringens in the proposed procedure are equal to or higher than recoveries in the existing procedures. In this work, we have not tested the selective inhibition of single strains of facultative anaerobes by the EY-free TSC agar. However, applications of this medium for enumeration of C. perfringens in naturally contaminated foods and in fecal specimens (A. H. W. Hauschild and R. Hilsheimer, manuscript in preparation) have shown essentially the same degree of selectivity as that of the egg yolk medium of Harmon et al. (6). Some shortcomings of the media containing egg yolk have been listed above: (i) the low selectivity of SFP agar; (ii) the relatively elaborate procedures; (iii) the frequent occurrence of C. perfringens colonies without discernible halos (false negatives); and (iv) the large and frequently spreading colonies which make 10fold dilutions impractical. We have also found that SFP agar allows growth of egg yolk-positive facultative anaerobes from foods. In some cases, these organisms produced completely opaque plates and thus masked the egg yolk reaction of C. perfringens. The lack of selectivity of the SFP agar has been overcome by replacing it with TSC agar (6). The remaining shortcomings of the egg yolk agars may be overcome by using EY-free TSC agar in pour plates and stab-culturing black colonies of supplemented NM agar for confirmation of C. perfringens. Comparison of D-CS from different sources. Since we did all of our work with D-CS from a single supplier (Nutritional Biochemicals Corp.), its effect on the enumeration of C. perfringens was compared with that of 1-CS from another company (Sigma Chemical Co., St. Louis, Mo.). Five C. perfringens strains were tested. Essentially the same results were obtained with -CS from both suppliers ( Table 4). Comparison of EY-free TSC agar with OPSP agar. Table 5 shows the recoveries of 22 C. perfringens strains in EY-free TSC and OPSP agars. As in preceding experiments (Tables 1 and 3), the recoveries of all strains were essentially quantitative in EY-free TSC agar. Twenty of these strains were also enumerated quantitatively in OPSP agar, but one of them (8247) produced only pin-point colonies that (9) by replacing its antibiotics with D-CS. The modified medium has not as yet been thoroughly tested.

v3-fos

2020-12-10T09:04:22.666Z

1974-08-01T00:00:00.000Z

237230197

s2

Storage of Stock Cultures of Filamentous Fungi, Yeasts, and Some Aerobic Actinomycetes in Sterile Distilled Water Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable. Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable. Several methods have been proposed for maintaining culture collections of fungi. Among these, dispersal of spores in sterile soil (1), sterile mineral oil overlays (3), deep freezing (4), ultra-low temperature freezing (9), and lyophilization (7) are the most favored. With the exception of the sterile soil and sterile mineral oil overlay techniques, the other methods involve time and expensive equipment. Castellani (5,6) reported maintenance of several cultures of human pathogenic fungi and yeasts in sterile distilled water for 12 months without any apparent changes in their morphology or physiology. A slightly modified version of Castellani's method, wherein physiological salt solution was substituted for distilled water and screw-capped bottles were used in place of cotton-plugged test tubes, was described by Benedek (2). Hejtmankova-Uhrova (8) reported successful maintenance of 73 strains of fungi belonging to 13 genera in sterile distilled water for 12 months. The present study comprises four independent experiments. They were initiated at different times to evaluate Castellani's technique of maintaining fungal cultures in sterile distilled water. The results of these experiments are presented. MATERIALS AND METHODS Four hundred and seventeen isolates of 147 species belonging to 66 genera were included. Of these genera, 48 were of filamentous fungi, 15 were yeast genera, and 3 were aerobic actinomycetes. The viability of fungal cultures stored in sterile distilled water was tested by four independent workers. Even though many of the species and genera investigated were common to all four experiments, the individual isolates selected by each worker differed in each experiment. Some of the genera were represented by several species, and each species in turn included several isolates. In some cases, on the other hand, the genus and species were represented by a single isolate. The first experiment was initiated in 1969, and covered only 16 isolates of 16 species belonging to 15 genera. The viability of these isolates maintained in sterile distilled water was tested only once, that is, after 60 months of storage. No attempt was made to revive these cultures between 1969 and 1974. Similarly, the second and third experiments were started in 1970 and 1972, and their results were read only once. The second experiment included 18 isolates of 17 species belonging to 12 genera. The third experiment covered 48 species belonging to 27 genera. Fifty-three isolates were included in the third experiment. The cultures stored in sterile distilled water from these two experiments were revived at the same time that those from the first experiment were cultured for viability. The fourth experiment, which was started in 1973, included a greater number of cultures than were used in the three previous experiments. One hundred and twenty-six species of 58 genera were represented by 330 isolates. These 330 water cultures were revived after 12 months along with the other cultures from the three previous experiments. Cultures were inoculated onto slants of potato dextrose agar in screw-capped tubes (20 by 150 mm) and were incubated at 25 C for 2 weeks. Six to seven milliliters of sterile distilled water was pipetted aseptically onto each 2-week-old culture. The spores and fragments of hyphae were dislodged by lightly scraping the aerial growth with the same pipette, and the resultant suspension was withdrawn and trans-218 ferred to a sterile glass 1-g vial. The cap of the vial was tightened to prevent evaporation of the water. The labeled vials then were stored at 25 C on laboratory shelves. incubated at 25 C for 3 weeks and were observed periodically for growth. Those cultures that did not grow by the end of 3 weeks were retested with the same procedure. When no growth was observed after the second subculture, the isolates were recorded as not viable. RESULTS AND DISCUSSION It became clear from the results of the four experiments (Tables 1 to 4) that the viability of the isolates ranged from 92 to 94%. None of the cultures were found to be contaminated by bacteria or other fungi. All the yeast cultures survived. In addition, several fungi that are notoriously difficult to maintain over an extended period, such as Aureobasidium pullulans, showed a better survival rate when stored in water than on conventional media. About 6 to 8% of the 417 cultures did not survive storage in water. These belonged to the following genera and species: Madurella mycetomi, Paracoccidioides brasiliensis, Trichophyton concentricum, T. gallinae, T. megninii, T. verrucosum, and T. yaoundii. All of these species are poor sporulators. On routine media like Sabouraud dextrose agar or potato dextrose agar, the above-mentioned species do not sporulate regularly. A reexamination of the storage vials of these species revealed that the inoculum had been too scanty in most cases. When inocula were adequate in regard to size, and consisted of several wefts of hyphae, even some of the poorly sporulating or nonsporulating species like T. violaceum, T. schoenleinii, and M. ferrugineum survived storage very well. To insure success, care must be taken to select actively sporulating isolates and to suspend adequate amounts of spores and pieces of hyphae in sterile distilled water for storage in vials. Care must also be taken not to allow the water to evaporate from the vials. Some of the revived cultures of species with perfect forms were tested for their mating ability after storage in water. The species like Arthroderma ciferrii and Petriellidium boydii readily formed ascocarps in abundance on oatmeal salts agar. In case of heterothallic species like Arthroderma uncinatum and Nannizzia incurvata, numerous ascocarps were formed on oatmeal salts agar when the revived cultures were crossed with the opposite mating type strains. In the case of other imperfect species, the revived cultures were comparable to the originals with respect to morphology and sporulation. The storage in water showed supression of pleomorphic changes in many cases, and, in cases of perfect species, showed no loss of mating competence. Results of the present study clearly show that the method of maintaining fungal cultures in sterile distilled water for extended periods of time is simple, inexpensive, and reliable. The method offers many advantages for laboratories that maintain small culture collections for reference or teaching purposes. The water culture technique is simple, as is the technique for reviving these cultures. The storage space required for the vials is minimal, and, since the vials are stored at room temperature, expensive refrigeration is not needed. The revival technique is less messy than that for cultures stored under mineral oil.

v3-fos

2018-04-03T01:23:41.726Z

1974-02-01T00:00:00.000Z

29355411

s2

Polygalacturonate lyase production by Bacillus subtilis and Flavobacterium pectinovorum. Nutritional factors relating to the production of polygalacturonate lyases by strains of Bacillus subtilis and Flavobacterium pectinovorum were examined. Studies were carried out in shake flask cultures. In the case of B. subtilis the enzyme was produced constitutively, whereas in the case of F. pectinovorum it was only produced in quantity in the presence of pectic substances. Glucose was the most suitable carbon source for production of the polygalacturonate lyase of B. subtilis; of the nitrogen sources examined, the highest activities per milliliter of supernatant and per milligram of cells were obtained with glutamine and ammonium sulfate, respectively. The pattern of enzyme production and growth was similar although enzyme production ceased at pH 5.3. Sodium polypectate was the best inducer of polygalacturonate lyase with F. pectinovorum. Highest activity per milliliter of cell-free supernatant was obtained with skin milk powder as nitrogen source, although ammonium sulfate gave highest enzyme production per unit of biomass. Growth of F. pectinovorum occurred between pH 5.7 and 7.2. Enzyme production occurred during active growth and was independent of the pH of the medium. Many species of wood are naturally resistant to treatment with preservatives, even when applied under pressure. During storage of Sitka spruce (Picea sitchensis) in water, bacterial degradation of the tori and bordered pit membranes takes place and this results in a marked increase in the permeability of the wood (1, 2). A number of bacterial species have been isolated from the sap of Sitka spruce poles that had been stored under water; two of these isolates were shown to possess high pectinolytic enzyme activity (4). This observation was significant because the pit membranes in the sapwood contain much pectic material. When these isolates were inoculated into sapwood blocks under laboratory conditions an increase in the permeability of the blocks occurred (3). The bacterial isolates have been identified as strains of Bacillus subtilis and Flavobacterium pectinovorum. Both isolates elaborate polygalacturonate lyase extracellularly and grow well and produce this enzyme in sapwood blocks in laboratory experiments. Polygalacturonate lyase activity has also been detected in sap, expressed from water-stored Sitka spruce (18). Polygalacturonate lyase was the only pectic enzyme produced by the bacterial isolates and was likewise the only pectinase detectable in expressed sap of water-stored Sitka spruce. The importance of pectinases in the process is indicated by the observation that a commercial pectinase was also capable of increasing the permeability of sapwood blocks (3). This investigation is a comparative investigation of some factors affecting production of polygalacturonate lyase by the isolates in batch culture. MATERIALS AND METHODS The basal mineral medium consisted of, in grams per liter: K2HPO4, 5.0 g; KH2PO4, 1.0 g; KCl, 1.0 g; MgCl,.6H20, 0.2 g; CaCl2 2H20, 0.1 g; MnSo4-4H20, 0.001 g; FeSO4.7H,0, 0.0005 g. Carbon sources, 0.5% (wt/vol), were added to this medium. The nitrogen sources consisted of complex nitrogen, (peptone, casein, etc.), 1.0% (wt/vol), amino acids 0.5% (wt/vol) or inorganic N, 0.2% (wt/vol). In studies on the effects of carbon and nitrogen sources on enzyme production, the initial pH (pHi) of the medium was 7.0 to 7.2. Media were dispensed in 50-ml volumes in 250-ml Erlenmeyer flasks, and autoclaved at 121 C for 15 min. A standard inoculum was prepared as follows: actively growing cells were centrifuged from the basal mineral medium containing bacteriological peptone (0.5%, wt/vol), washed, and suspended in sterile saline to give an optical density (OD) reading of 10.0 in an EEL spectra colorimeter at 600 nm. All media were inoculated with 1.0 ml of this standard suspension and shaken at 150 rpm in a New Brunswick orbital incubator (model G25) set at 27 C. VOL. 27,1974 LYASE PRODUCTION BY B. SUBTILIS AND F. PECTINOVORUM Enzyme activity was measured in the cell-free supernatant (CFS). In studying the effect of pH on growth and enzyme production, biomass and enzyme activity estimations were carried out throughout the growth cycle of the organism. In other investigations, enzyme activity was assayed when enzyme production had ceased in the stationary phase of growth. Variations in activity between duplicate culture flasks were negligible, and results were reproducible on repetition of experiments. Acid-soluble pectic acid (ASPA) was prepared by the method of McCready and Seegmiller (11). Polygalacturonate lyase activity was measured with a solution containing 0.2% (wt/vol) ASPA, 0.001 M CaCl2, and 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 8.0. The substrate was prepared immediately before use. Appropriately diluted, cell-free supernatant (0.1-ml samples) was added to 2.0 ml of substrate in all assays. The rate of change in absorbance at 235 nm was measured in a 1-cm cell, using a Pye Unicam SP 500 spectrophotometer equipped with a constant temperature unit. An enzyme unit is defined as the lyase activity releasing 1 Mmol of product per min at 30 C. A molar absorbtivity of 4,600 M-'/cm was used for this calculation (16). Activities are expressed in units per milliliter of CFS. However, to establish that differences in enzyme activities were not merely due to differences in bacterial growth, activity was also determined as a function of biomass (i.e., in units per milligram of biomass). This value provides a better measure of enzyme induction. Biomass was measured in OD units at 600 nm in an EEL spectrophotometer. OD units were converted to milligrams (dry weight) of cells by reference to a standard curve. Results of biomass determinations are expressed as milligrams per milliliter of CFS. When an insoluble carbon source was used in the growth medium, biomass was not measured. RESULTS Enzyme production by B. subtilis. The effect of carbon source on polygalacturonate lyase production by B. subtilis is summarized in Table 1. A 0.2% (wt/vol) amount of (NH4)2SO4 was added as nitrogen source to the basal medium. Although considerable enzyme activity was produced on many carbohydrates, glucose was clearly the best carbon substrate. Saccharides containing two or more glucose units were the poorest inducers. Glucose was incorporated as carbohydrate in media used to investigate the effect of nitrogen source on enzyme production. The results are presented in j'able 2. Although highest enzyme activity per milliliter of CFS was measured in media containing glutamine, highest induction as related to biomass was detected with (NH,)2SO as nitrogen source. However, significant levels of enzyme activity were measured with all nitrogen sources. The effect of pH on growth and polygalacturonate lyase production by the B. subtilis isolate is illustrated in Fig. 1. (NH4)2SO4 and glucose were incorporated into the basal salts medium. Although bacterial growth occurred between pH 4.7 to 7.5, the growth rate decreased with increase in pH. Because growth was accompanied by acid production, growth stopped when the pH dropped to about 4.5. Generally, the pattern for enzyme production was similar to the growth pattern. However, production stopped when the pH of the medium dropped to 5.3, although growth was not impaired. Enzyme production by F. pectinovorum. In determining the effect of carbon source on polygalacturonate lyase production by F. pectinovorum, (NHj2SO4 was added as nitrogen source to the basal medium. The results are presented in Table 3. The enzyme was only induced by pectic substances. On other carbohydrates only a very low level of activity was observed. Sodium polypectate was added as inducer to media used in investigating the effect of nitrogen source on enzyme elaboration by this organism (Table 4). While highest activity per milliliter of CFS was obtained when skim milk was used as a nitrogen source, activity reflected the high biomass production. The carbohydrate content of skim milk made little or no contribution to the level of activity produced (Table 4). (NH4)2SO4 caused highest enzyme production per unit of biomass. Apart from skim milk, enzyme production on complex nitrogen sources was completely inhibited or low ( Table 4). The influence of pH on growth and enzyme APPL. MICROBIOL. production is illustrated in Fig. 2. Sodium polypectate and (NH,)2SO were added to the basal medium in this experiment. Bacterial growth, which in this case was accompanied by a slight rise in pH, occurred between pH 5.7 and 7.2. Within this range the growth rate increased with increase in pH. Enzyme production occurred during active growth by the organism. Although enzyme production was affected by the growth rate, it appeared to be otherwise independent of the pH of the medium. DISCUSSION Although both organisms perform a similar function in increasing wood permeability, the above study reveals that the polygalacturonate lyase is constitutively produced by the B. subtilis isolate but is only induced in F. pectinovorum when the medium contains pectic substances. Apart from F. pectinovorum, inducible polygalacturonate lyase has been observed with Erwinia spp. (21), Pseudomonas fluorescens (5,7,20), Pseudomonas marginalis (13), xanthomonads (17), Xanthomonas campestris (14), and Clostridium multifermentans (9). On the other hand, although constitutive polygalacturonate lyase synthesis was observed with Aeromonas liquefaciens (8) and with Erwinia spp. (12), in contrast to B. subtilis, activity was repressed by glucose. Repression of constitutive enzyme production by glucose is a very common phenomenon (D. D. Brown and J. Monod, Fed. Proc., 20:222, 1961; 10). In a number of rumen bacteria (19) and in a strain of P. marginalis (21), constitutive polygalacturonate lyase production was not repressed by glucose. With both B. subtilis and F. pectinovorum, different degrees of repression were observed when nitrogen sources other than (NH,)2SO were added to the medium. In contrast to this, Hancock et al. (6) observed that polygalacturonate lyase elaboration by Hypomyces solani was reduced when individual amino acids or inorganic nitrogen sources were substituted for casein hydrolysates in the growth medium. As in the case of B. polymyxa, polygalacturonate lyases of F. pectinovorum and B. subtilis are produced mainly in the log phase of growth. The lyases of these two isolates are classical extracellular enzymes, according to the criteria established by Pollock (15), in that they are produced during active growth, are found in the cell-free supernatant, and are secreted before extensive cell lysis has occurred. The pH range for growth of B. subtilis was wider than that of F. pectinovorum. Enzyme production occurred in F. pectinovorum over its complete growth range, but not at lower pH values in the case of B. subtilis. Optimal elaboration of polygalacturonate lyase by H. solani (6) occurred within the pH range for production of this enzyme by the two isolates. During water storage, the pH of the sapwood generally varied from 7.0 to 5.0 (2). It has been illustrated that both organisms grow and elaborate the enzyme within this pH range. The growth rate of the B. subtilis isolate was higher at lower pH values, whereas F. pectinovorum had an increased growth rate at higher pH values. This study is part of a research project undertaken to investigate the possibility of utilizing artificial water storage tanks, seeded with selected bacteria, to increase permeability of softwoods to treatment with preservative. The high pectinolytic activity and resultant ability of the isolates to increase sapwood permeability renders them suitable for use in commercial storage plants for increasing wood penetrability of preservatives.

v3-fos

2020-12-10T09:04:20.449Z

1974-10-01T00:00:00.000Z

237233364

s2

Production of Patulin by Penicillium expansum Twenty-seven isolates of Penicillium expansum Lk. ex Thom obtained from Europe, Australia, and North America from seven different fruit hosts all produced patulin in culture. Six isolates were essentially nonpathogenic in apple fruits. In culture, patulin generally accumulated to much higher levels than in apple fruits. At all temperatures permitting fungus growth, patulin was produced. However, only small amounts were observed near the maximal temperature for growth (30 C). At 0 C, patulin accumulated but slowly in culture. Modified atmospheres suppressed both fungus growth and patulin accumulation in apples. After varying incubation periods to obtain similar total growth, the patulin concentration was low in modified atmospheres and high in air. The mycotoxin patulin is produced by several fungi, mostly species of Aspergillus and Penicillium. Included is Penicillium expansum, the blue-mold fungus of deciduous fruits. Its ability to grow at 0 C makes it a major cause of loss of apples and pears in storage. Other common fruit hosts include most Prunus sp., grapes, and persimmons. Although patulin is acutely toxic to humans, its acute toxicity is believed unimportant because rotted flesh is easily avoided in fresh fruits and rotted fruits are eliminated before processing, thereby limiting ingestion. Longterm health problems from patulin-contaminated foods were suggested, however, when rats developed sarcomas after subcutaneous injections (5), although malignancies have evidently not resulted from oral ingestion (6,11). Because patulin is heat stable (8), normal heat processing of fruits does not destroy it. Patulin has been found in apple fruits (2,7) and in apple juice (16,17). Although Brian et al. (2) reported that some apple lesions did not contain patulin, Wilson and Nuovo (17), using a more sensitive detection procedure, found patulin in all of 60 cultures tested (all isolated from apples produced in a single orchard in Vermont). Most studies of patulin production have utilized isolates from apples. Patulin production possibly varies considerably among isolates from different hosts and widely separated regions. This study was done, therefore, to compare patulin production in vitro and in vivo by isolates from different fruit hosts and widely separated regions. An additional objective was to determine the effects on patulin production of the temperatures and modified atmospheres used in fruit storage and transit. MATERIALS AND METHODS Cultures. Except as indicated otherwise, we isolated fungi from blue-mold lesions of deciduous fruit hosts and at locations shown in Table 1. Cultures were maintained on potato-dextrose agar slants stored at 7 C. To determine their capacity to produce patulin in culture, each isolate was grown in potato-dextrose broth prepared from fresh potatoes. After 600 ml of medium was dispersed in cotton-plugged, 2.4-liter reagent bottles, it was autoclaved for a time (60 min at 121 C) sufficient to produce a golden yellow color as suggested by Norstadt and McCalla (13). After inoculation, the bottles were placed on their sides and incubated at 22 to 24 C without agitation. Portions (15 ml) of broth were withdrawn aseptically from beneath the mycelial mats after 6,9,14,16,20, and 23 days and extracted. Patulin was estimated by inhibition of Bacillus megaterium as described below. The pathogenicity of the isolates to Golden Delicious apple fruits was determined by stabbing opposite sides of each fruit to a depth of 2 to 4 mm with a needle covered with conidia. Fruits were incubated at 23 C in nonsealed polyethylene bags humidified by insertion of several wet paper towels. Relative pathogenicity was related to diameter of lesions after 6 days. Patulin levels in disease lesions were determined at 10 days of incubation after inoculation as above. Fungus-invaded tissue of lesions was removed and weighed, and patulin was extracted as indicated below. Extraction. Patulin was extracted from 15-ml portions of culture broth by adding an equal volume of ethyl acetate, hand shaking for 2 min, centrifuging for about 2 min to hasten phase separation, and decanting the ethyl acetate. The extraction was twice repeated, and the ethyl acetate fractions were combined and dried over anhydrous MgSO4 for 20 to 30 589 min. After filtering under slight vacuum, the extracts were evaporated to dryness under N2, and the residue was dissolved in CHClI and adjusted to a convenient volume for analysis. Patulin extraction from apple tissue utilized a modification of the method of Scott and Somers (15), who reported a 75 to 90% recovery of patulin from fruit juice. In brief, 54 g of lesion tissue plus 50 ml of water were blended, and the slurry was transferred to centrifuge tubes and extracted thrice as described above. After filtering, the combined ethyl acetate fractions were passed through a chromatographic column (2 x 40 cm) packed with 25 g of silica gel (60 to 100 mesh). The column was washed with about 100 ml of ethyl acetate. All the eluate was collected, evaporated to dryness under vacuum, dissolved in CHCl,, and adjusted to 1.0 ml. Patulin was first estimated by comparing the quenching of fluorescence under short-wave ultraviolet light with that of authentic patulin. Subsequently, the phenylhydrazone derivative was prepared by spraying the plates with 3% NH4OH followed by 4% aqueous phenylhydrazine and heating for 2 to 3 min at 110 C. Biological activity at the patulin R, was sometimes verified by scraping that area from thinlayer chromatography plates after ultraviolet examination. After extraction with CHCl,, the patulin was transferred to a 6-mm disk prepared from Whatman 3MM paper and assayed by inhibition of growth of B. megaterium, a technique suggested by Clements (4) for assay of aflatoxins. Inhibition zones were compared with a standard curve previously established with authentic patulin. Diameters of zones of inhibition were near a straight line between 1 and 60 fig when concentrations were on the logarithmic scale of a semilog plot and diameter was on the arithmetic scale. Temperatures. To determine the effect of temperature on mycelial growth and patulin accumulation, isolate 17 was grown in potato-dextrose broth. A sample (50 ml) of medium was autoclaved for 60 min at 121 C in cotton-plugged 125-ml Erlenmeyer flasks. After inoculation, the flasks were incubated at 23 C for 24 h before transfer to constant-temperature rooms (± 1 C). Forty flasks were incubated without agitation at each of the following temperatures: 0, 5,10,15,20,25, and 30 C. At intervals of 3 days to 2 weeks, depending on the temperature, 15-ml portions of broth were removed from each of two flasks for patulin analysis as described above. The mycelial mats were transferred to tared Buchner funnels and washed with the aid of vacuum. After drying for 24 h at 60 C, growth was recorded as milligrams (dry weight)/per milliliter. Modified atmospheres. Air or an artificial atmosphere was passed through a 4-liter glass jar containing five apples inoculated with P. expansum isolate 17 or 24 as described previously. A flow rate of 250 to 300 cm3/min prevented appreciable atmosphere changes due to fruit respiration. Artificial atmospheres were prepared by mixing flowing air with tank CO2 and/or N2. Gas flows were controlled with capillary flow meters as described by Claypool and Keefer (3). RESULTS AND DISCUSSION Concentrations of extractable patulin varied from less than 10 jig to nearly 1 mg/ml when cultures were grown in vitro (Table 1). Patulin concentrations were much lower in the watersoaked tissue of fungus lesions than in culture, varying from 2 to 125 ug/g (fresh weight). No isolate of P. expansum failed to produce patulin. Isolates that accumulated the highest quantities in vitro were not necessarily the highest when grown in vivo. For example, isolate 3 accumulated only 2 jug/g in vivo versus 330 gg/ml in culture. On the other hand, isolate 27 accumulated less than 10 Ag/ml in culture but had a concentration of 50 ,g/g in apple tissue. In addition, six isolates obtained from fruits other than apple were so weakly pathogenic in apples that no in vivo determinations were possible. Observations that patulin is a phytotoxin, being implicated in apple replant problems (1) and poor growth of seedling wheat (12), suggest that it may play a role in the pathogenicity of P. expansum to deciduous fruits. In our tests, however, patulin concentrations in vivo did not relate closely to pathogenic vigor. The most vigorous pathogens were isolates 7 and 8, from Queensland and Michigan, respectively, but the lesions contained only 7 ttg/g. On the other hand, isolates 6, 11, 12, and 16 contained 80 Ag/g in lesions but were only about average in pathogenicity. In studies on the influence of temperature, growth of P. expansum (isolate 17) reached a maximal dry weight of 0.17 g/flask (3.7 mg/ml) at all temperatures ( Fig. 1 and 2). This maximum was reached in 2 to 3 weeks at 20 to 30 C but required 16 weeks at 0 C. Patulin accumulated to about 0.6 to 0.7 mg/ml in flasks incubated at 0 to 20 C. At 30 C, near the highest temperature permitting fungus growth, the maximal fungus growth was attained within 3 weeks, but patulin never exceeded 0.07 mg/ml. Hence, 30 C is presumably much less favorable for patulin accumulation than for fungus growth. At 20 and 25 C, patulin levels decreased 14.0 strikingly after reaching peak levels ( Fig. 1), suggesting a metabolic destruction. On the other hand, Pohland and Allen (14) showed that patulin gradually disappeared in aqueous solutions. In limited tests, Gravenstein apples were inoculated with conidia of isolate 17. Patulin levels were compared in similar-sized lesions that had developed at 20 and at 0 C. After 5 days at 20 C, lesions were about 2 cm in diameter at the fruit surface and contained about 300 ,ug of patulin per g of fresh apple tissue. At 0 C, 36 days were required to obtain lesions of the same size that contained a maximum of about 100 ug/g. It is thus evident that storage at 0 C does not prevent patulin accumulation in apple fruits. Modified atmospheres containing high concentrations of CO2 (20 to 60%) reduced growth of Penicillium martensii and penicillic acid production in corn (10). High levels of CO2 (20 to 100%) or low concentrations of 02 (<5%) dramatically reduced growth of Aspergillus flavus and aflatoxin accumulation in peanuts (9). Because growth of P. expansum is also suppressed by atmosphere modifications, we tried to separate growth effects from patulin production. Incubation periods were varied to obtain disease lesions of approximately equal size (3.5 cm in diameter). Therefore, the increased incubation period in modified atmospheres to develop a lesion of that size is a measure of growth suppression (Table 2), which varied considerably among the isolates used. Either elevated CO2 or reduced 02, within the range tolerated by most living fruits (2 to 8% CO2 and 2 to 3% 02), suppressed patulin levels. Thus, patulin suppression was in addition to growth suppression. Since both growth and patulin accumulation are therefore suppressed, use of modified atmospheres in storage and transit might reduce the patulin hazard in deciduous fruits.

v3-fos

2020-12-10T09:04:12.824Z

1974-05-01T00:00:00.000Z

237233220

s2

Inactivation of Dried Bacteria and Bacterial Spores by Means of Gamma Irradiation at High Temperatures Dried preparations with Streptococcus faecium, strain A21, and spores of Bacillus sphaericus, strain CIA, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodurans, strain R1, and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resistant sporeformer B. sphaericus, strain CIA, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment. resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment. It has been suggested that combinations of heat and ionizing radiation might be useful for sterilization of interplanetary vessels and foodstuffs (5,7). Such combinations may act additively or synergistically in inactivation of microorganisms without correspondingly additive or synergistic effects in material damage. Items which do not tolerate a dry heat sterilization or a radiation sterilization may thus be sterilized by a combination of the two methods. The present work aims at an evaluation of gamma irradiation procedures combined with heat treatments for sterilization of medical equipment. We have applied the bacteriological standard preparations we normally use for control of radiation sterilization and dry heat sterilization of such equipment supplemented with some experiments on a few other radiationresistant or heat-resistant strains. MATERIALS Test pieces with S. faecium, strain A11, were taken from batches of reference standards prepared for control of the microbiological efficiency of radiation sterilization plants (3). Briefly, these standard preparations are prepared as follows. Heavily inoculated 5% blood agar plates are incubated at 30 to 32 C for 3 to 4 days. The cultures are scraped off the plates and suspended in serum broth. Droplets (0.02 ml) of the suspension (containing around 10' viable units) are placed on polyethylene foil and dried overnight in a glove box at 30% relative humidity as it has been found that humidity levels above 50% lead to reduced radiation resistance of the test pieces (4). Each dried test piece is sealed into a double envelope of polyethylene foil. Viable counts are determined by means of classical dilution techniques, by using 5% blood agar plates and by incubation at 30 to 32 C for 3 to 4 days. Test pieces with M. radiodurans, strain R,, were prepared by similar procedures. The strain was cultivated on tryptone-glucose-yeast extract (TYG) agar, and the test preparations were dried at ambient humidity in a laminar air flow cabinet. Test pieces with spores of B. sphaericus, strain CIA, were taken from batches of spore monitors pre-83() pared for control of the microbiological efficiency of radiation sterilization plants (3). These test pieces are produced by procedures similar to those used for preparing test pieces with S. faecium, strain A,1. They are dried at ambient humidity. For experiments at 100 C the test pieces were removed from the polyethylene envelopes and transferred to small aluminum capsules. Test pieces with spores of B. subtilis were taken from batches prepared for control of sterilization by dry heat, formalin, and ethylene oxide (6). Briefly these standard preparations are prepared as follows. Plate cultures from agar plates incubated for 5 days at 37 C are scraped off the plates and suspended in physiological saline and then mixed with sterilized quartz sand. The mixture is immediately vacuumdried at a pressure of 2 to 4 mm Hg for 24 h, and finally homogenized in a mortar. Each test piece, which consists of 120 mg of this spore sand and contains 2 x 10' viable units, is wrapped in two layers of paper. Spores of B. globigii were used in two kinds of preparations: dried serum broth preparations prepared in the same way as test pieces with spores of B. sphaericus, strain C A, and sand preparations prepared in the same way as test pieces with spores of B. subtilis. Most of the gamma irradiations were carried out in "Co-facility II, a gamma cell, which at the time of the experiments was loaded with 600 curies. During the time period, where experiments were undertaken, the dose rate in the center of the irradiation chamber decreased from 75 krad/h to 64 krad/h. Irradiations under this condition will be referred to as 70 krad/h. The dose rate could be reduced by lead shieldings. Two shielding configurations were -used. In one of these configurations the dose rate was reduced by a factor of 2.8, and irradiations under this condition will be referred to as 25 krad/h. In another configuration the reduction factor was 4.45 or 16 krad/h. For simultaneous heating and irradiation test, pieces were placed in the gamma cell in a 2-cm-wide brass cylinder, which was surrounded by a heat jacket. The temperature in the center of the cylinder was measured by an electrical thermocouple. The temperature varied around the setting with i 2 C. In experiments where heating was applied before or after irradiations, irradiations were carried out in ""Co-facility m, loaded with 4,500 curies. The dose rate in the center of this gamma cell was 400 krad/h. RESULTS Radiation inactivation curves for dried serum broth preparations with S. faecium, strain A21, M. radiodurans, strain R., spores of B. sphaericus, strain C A, and spores of B. globigii irradiated at room temperature are seen in Fig. 1. Figure 2 presents radiation inactivation curves for the same preparations at 80 C and 25 krad/h. It will be seen that the inactivation curves for the two spore preparations were unchanged, but that the LD 99.99 value for the preparations with the two vegetative strains was reduced by about a factor four. (LD 99.99 is the radiation dose, or heating time, that reduces the surviving fraction to 10-4, in Fig. 2: 0.8 Mrad for S. faecium, strain A21.) When the standard preparations with the vegetative bacteria were kept at 80 C for 48 h the surviving fraction for both strains was higher than 10%1, SO the simultaneous application of dry heat at 80 C and gamma irradiation acted clearly synergistically in the experiments with these two vegetative strains. At 80 C and 70 krad/h, the LD 99.99 was 1.4 Mrad for S. faecium, strain A21, and 1.6 Mrad for M. radiodurans, strain R1. At 60 C and 70 krad/h, the LD 99.99 for strain A21 was 2.8 Mrad and for strain R, it was 3.4 Mrad. Table 1 presents LD 99.99 values for irradiation of the four kinds of spore preparations at room temperature and at 100 C. It can be seen that the radiation resistance of the test pieces with spores of B. subtilis, normally used for control of sterilization by dry heat, was the same at 100 C and room temperature at the three dose rates tested. The radiation resistance of test pieces with spores of B. sphaericus, strain C IA, normally used for control of radiation sterilization plants was lower at 100 C than at room temperature. At 70 krad/h the reduction was small; at 16 and 25 krad/h the resistance was reduced by about a factor of four to the level of resistance observed with the subtilis preparations. The radiation resistance of the two kinds of preparations with spores of B. globigii at 100 C was lower than the resistance of the preparations with spores of B. subtilis and B. sphaericus at the same irradiation conditions. The radiation resistance of spores of B. subtilis and B. globigii in sand preparations was close to the resistance reported for clean spores of these strains (2,7). The radiation resistance of the B. globigii spores in serum broth preparations at room temperature was within the range reported for similar preparations with spores of B. subtilis and B. globigii (1,2). A few experiments with application of heat (100 C) and irradiation (400 krad/h) separately were carried out with preparations of spores of B. sphaericus, C IA. The LD 99.99 for heat alone was 100 h. When spores were pre-irradiated with 0.6 or 1. Fig. 1 irradiated at 80 C with 25 krad/h. DISCUSSION In the experiments with irradiation at 80 C, the two vegetative strains had a reduced radiation resistance, whereas the spores of B. sphaericus and B. globigii in serum broth preparations had an unchanged response. There were no indications of the so-called paradoxical effect, which has been reported with spores of B. megaterium and Clostridium botulinum (5,8). In these reports the spores had a higher radiation resistance at 80 to 90 C than at room temperature. This paradoxical effect disappeared at 100 C where the radiation resistance dropped to below the level observed at room temperature. In cases where it is known that sporeformers are totally absent, it may be beneficial to increase the irradiation temperature to 80 C in order to reduce radiation doses, but if the occurrence of sporeformers cannot be excluded, there may be a limitation of any benefits by irradiation at this temperature. At 100 C the spore monitor for radiation sterilization B. sphaericus, strain CIA, was the most resistant at all conditions tested. This, however, does not indicate per se that these preparations can be regarded as ideal monitors for evaluation of any combination of radiation and heat for sterilization, as more suitable strains may be isolated from the relevant environments. However, the present data may be applied in a preliminary analysis aiming at an identification of combinations of radiation and heat which may warrant further consideration, excluding combinations where benefits are too small to warrant investments in microbiological research and technical development. Figure 3 represents an attempt to do such an analysis based on the data obtained with B. sphaericus, strain CIA, at 100 C. In this analysis we arbitrarily have demanded that any combination of heat and radiation, which may warrant further consideration, at least should reduce both radiation LD 99.99 values and heat LD 99.99 values with a factor of two. The ordinate in Fig. 3 is LD 99.99 in hours, the abscissa is the dose rate in krad per hour. In this scheme we have drawn a full line, which represents the LD 99.99 values one would obtain if radiation and heat acted additively. The experimental results obtained at the three dose rates tested are plotted for comparison. It will be seen that at 16 and 25 krad/h the combination of heat and radiation acted more effectively than a pure additive effect, and that the results at 70 krad/h correspond to an additive effect. It can be seen that the only set of experimental data, which was below both "half" lines, were the data obtained at 25 krad/h. At this point the LD 99.99 values for both heat and radiation were reduced with about a factor of three. At 16 krad/h the reduction in radiation LD 99.99 value was the same as at 25 krad/h, whereas the heat value was only reduced by a factor of two. At 70 krad/h the data were below the "half' line for heat, but above the line for radiation. It appears from this analysis that combinations of heat and irradiation, where halving of both sterilization time and dose may be expected, are concentrated to a narrow dose-rate interval, approximately 20 to 40 krad/h. It appears unlikely that sterilization times and doses may be reduced by more than a factor of three at any combinations of 100 C heat and irradiation. Technological and economic considerations must decide whether the possibility of introducing this combination method in medical sterilization can justify the amount of microbiological work that will be necessary in order to ensure the general microbiological safety of the process. In view of the data presented here the sterilization times and doses suggested by Reynolds and Garst (7) for sterilization of spacecraft appear to be about one order of magnitude too low for sterilization of equipment for medical use. Although they use a slightly higher temperature (105 C), their suggestions of 132 krad at 12 krad/h or 252 krad at 36 krad/h, for example, as possible sterilization process parameters appear to be very optimistic. With any of the four kinds of spore preparations we have tested at 100 C, irradiation with 250 krad gave inactivation factors smaller than 10'.

v3-fos

2020-12-10T09:04:17.243Z

1974-04-01T00:00:00.000Z

237231723

s2

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 103 spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 108 spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F. 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10' spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10' spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F. Clostridium botulinum type E was first reported by Gunnison et al. in 1935 (4). The strains studied were isolated by Kushnir (7) in Russia from the intestines and muscles of sturgeon. At about the same time, E. Hazen (5,6) isolated two new strains from outbreaks of botulism caused by smoked salmon and canned sprats, and it became evident that her isolates were type E. Subsequently, numerous outbreaks of type E botulism have occurred in many parts of the world with fish, fish products, or other marine species identified as the major sources of poisoning. In the United States the organism has been isolated from marine muds, waters, and marine animals from the Pacific Northwest (3), the Gulf of Mexico (14), the Atlantic Coast (15), Lake Cayuga (2), and the Great-Lakes (1). The ability of C. botulinum type E spores to germinate, multiply, and produce toxin at refrigeration temperatures has been well documented. In 1961 Schmidt et al. (11) showed toxin production by four type E strains in a beef stew medium at 38 F after 31 to 45 days. Lerke and Farber (8) tures apparently is substrate dependent; the more suitable the substrate the lower the temperature at which spores will germinate and produce toxin. This paper describes type E botulinum toxin production in meat of the blue crab, Callinectes sapidus, at temperatures of 40 F and above and the effects of a recommended minimal pasteurization (185 F for 1 min) upon varying concentrations of C. botulinum type E spores in crabmeat. Reported also are the results of a brief survey to determine the incidence of type E spores in bottom mud samples taken from areas where crabs are obtained for processing plants in Crisfield, Md. MATERIALS AND METHODS Cultures. Four strains of C. botulinum type E were used, no. 17786 from the American Type Culture Collection, and Beluga, 070, and G21-5 strains from the Food and Drug Administration, U.S. Department of Health, Education, and Welfare. Antisera. Types A, B, and E antisera were supplied by Matteo Cardella at Fort Detrick, Frederick, Md. An additional supply of types A and B antisera was obtained from W. S. Hammond of Lederle Laboratories, Pearl River, N.Y. For toxin typing, antisera A and B were diluted 1:10 and antisera E was diluted 1:100 in 0.85% saline solution. Media. Cooked meat medium (Difco) was used for culture maintenance and for determination of the most probable numbers (MPN) of viable spores. The spore production medium consisted of 5% Trypticase, 0.5% peptone, 0.4% glucose, 2% yeast extract, and 0.1% sodium thioglycollate (TPGY). TPGY containing 0.1% filter-sterilized trypsin (TPGYT), an improved medium for the isolation of type E reported by Lilly et al. (9), was used to detect type E spores in the mud samples. Gel-phosphate buffer (0.2% gelatin in 0.4% Na2HPO4 in distilled water) at pH 6.2 served as a diluent for toxin and spores. Mud samples. Mud samples were taken from those areas of the Chesapeake Bay adjacent to Crisfield that were being actively fished with crab pots. Bottom mud samples, taken with a Ponar grab sampler, were placed in cans with press-type lids and stored at 36 F until they were examined. For detection of type E spores, 15 g of well-mixed mud were placed in wide-mouth bottles containing glass beads and 15 ml of gel-phosphate diluent. The bottles were shaken vigorously and then allowed to settle for 10 min. The supernatant was decanted into flasks of TPGYT and incubated for 3 to 5 days at 85 F. Broth (10 ml) was then transferred to a screw-capped test tube and frozen for toxin typing. For detection of heat-labile toxin, 0.5 ml of a 1: 10 dilution of unheated culture fluid was inoculated intraperitoneally into mice weighing 19 to 24 g, and a second group of mice was inoculated with a 1: 10 dilution of growth fluid that had been heated for 10 min in boiling water. The mice were observed for 48 h. Survival of the mice receiving heated sample and death of those receiving unheated sample suggested the presence of botulinum toxin. Botulinum toxin was confirmed and typed with specific antisera. The toxic broth was thawed, and separate 0.25-ml portions of a 1: 10 dilution were mixed with 0.25 ml of type A, B, or E antiserum. The mixtures were held at room temperature for 1 h and then inoculated into mice. Spore preparation. The type E stock cultures were inoculated into cooked meat medium and incubated at 85 F for 24 h. TPGY medium (500 ml) was inoculated from the 24-h cooked meat tube and incubated at 85 F for 5 to 7 days. The broth was then centrifuged for 30 min, and the sediment was suspended in gel-phosphate diluent at 2% of the original volume. The concentrated spore suspensions were centrifuged and washed twice in diluent, and the MPN of viable spores was determined by inoculating serial 10-fold dilutions into cooked meat tubes, three tubes per dilution. In all MPN determinations, trypsinized culture fluids from those positive growth tubes defining the end point were inoculated into unprotected mice and mice protected with type E antiserum for toxin identification. Growth in crabmeat and crabmeat homogenate. Freshly picked crabmeat was packed tightly into screw-capped test tubes, approximately 10 g per tube. Crabmeat homogenate, prepared by blending 1 part crabmeat with 2 parts 1% saline in a Waring blender for 90 s, was dispensed in 10-ml samples to screwcapped test tubes. The tubes with whole meat and homogenate were autoclaved for 15 min at 250 F, cooled rapidly in cold water, and inoculated with 0.1 ml of spore suspensions. Inoculum per tube was up APPL. MICROBIOL. to a maximum of 106 spores. The inoculated tubes with caps loosened were placed in Gas Pak (BBL) jars to assure anaerobiosis and incubated at 85 F, room temperature (approximately 75 F), 50 F, and 40 F. The tubes were examined periodically for gas production, off odors, and for toxin by mouse inoculation. The procedures used for detection of toxin followed those outlined in Chapter 2 of the Bacteriological Analytical Manual (13). Whole meat was homogenized with an equal weight of gel-phosphate diluent and centrifuged to separate the extract. Similarly, the contents of the crabmeat homogenate tubes were compressed, and extract was obtained by removal of the separated liquid. Filter-sterilized extracts were mixed 1: 1 with 1% trypsin, held for 45 min at 37 C, and inoculated into mice without further dilution. Toxin type was verified by inoculation of the trypsinized extracts into mice protected with 0.5 ml of type E antiserum. Pasteurization experiments. One-pound (373.24-g) quantities of crabmeat were inoculated by thorough mixing with 4.5 ml of spore suspensions adjusted to give an inoculum range of from 106 to 108 spores per 100 g of meat. The inoculated meat was packed into one-pound cans (401 x 301), hermetically sealed, and heated in a 190 F water bath. One can of crabmeat in each pasteurization run was fitted with a thermocouple passing through the side of the can to the geometric center for determination of internal meat temperatures. The thermocouple was connected to a potentiometer from which the temperature readings were made. Those cans receiving full pasteurization remained in the water bath until the internal meat temperatures had reached 185 F. They were held at 185 F for 1 min and transferred to an ice bath for rapid cooling (12). When the internal meat temperatures had dropped to 100 F, the cans were moved to refrigerated storage at 40 F. During four of the six pasteurization runs, three cans were removed at regular intervals from the hot water bath. These were immediately tested for type E spores by the MPN method. Of processed cans stored at 40 F, one from each pasteurization run was removed monthly and tested for toxin. RESULTS Incidence of C. botulinum type E in crab fishing areas. The results of the survey are shown in Table 1. Twenty-four samples were analyzed, and C. botulinum type E was detected in 21. The presence of type E was suspected in the other three since they contained a heat-labile toxin that killed mice within 16 h, but the results of toxin typing were inconclusive in that the mice were protected by both A and E antisera. Possibly, the broth contained both types of toxin, each at a level too low to kill the mice independently. Growth and toxin production in crabmeat and crabmeat homogenate. Over a 2-month period at room temperature, only 2 out of 84 tubes of autoclaved whole crabmeat inoculated with 2.4 x 105 spores became toxic after 12 and 17 days. The reason for the low incidence of toxin development in the whole meat is not understood. Perhaps autoclaving altered the substrate in some way, adversely affecting growth or toxin production by type E spores. Four tubes of unautoclaved crabmeat inoculated at the same spore level spoiled rapidly at room temperature, and after 3 days all four contained type E toxin. It is suggested that the growth of the natural flora in these tubes produced anaerobic conditions necessary for growth and toxin production by the type E spores. Table 2 shows the number of days incubation at 50, 75, and 85 F required before toxin could be detected in homogenates inoculated with 100 type E spores. The same time periods were required for toxin development in homogenate containing as little as a calculated five spores per tube. Below five spores, not all homogenates became toxic, and toxin development was usually though not always delayed by 24 to 48 h. At 50 F and above, toxin development was invariably accompanied by gas production and a slight sour odor which could easily remain undetected. Toxin development in homogenates at 40 F was considerably delayed, and more highly concentrated inocula were required as shown in Table 3. Toxin was not detected in any tube receiving less than 1,000 spores. In the majority of cases, there was no visible gas production nor were any odors detectable to indicate growth and toxin formation by the type E spores. Pasteurization experiments. The six pasteurization tests are summarized in Table 4. The data show the surviving spores when meats containing 106 to 10' spores per 100 g are given the recommended minimal pasteurization of 1 min at 185 F. All samples remained free of toxin during 6 months of storage. No storage data are reported for pasteurization no. 6, since all the cans were used to determine the MPN of surviving spores during the pasteurization cycle. In pasteurizations three through six the Beluga strain was used since it was the only spore suspension sufficiently concentrated to deliver spore levels of 107 per 100 g and above. The recommended minimum pasteurization process for crabmeat by the water bath method is, by definition, the heating of hermetically sealed containers in a 190 to 192 F water bath to an internal meat temperature of 185 F at the geometric center of the container and holding for 1 min at that temperature (12). The lethality of the process is determined by the cumulative effect of time and temperature; therefore, the come-up time to pasteurization temperature contributes significantly to the effectiveness of the over-all process. The relationship of time and temperature to spore survival throughout the pasteurization cycle is shown by the data in Table 5. Although an initial temperature of 60 to 65 F is recommended (12), a range of initial temperatures from 33 to 66 F did not significantly affect the final spore survival. DISCUSSION Fresh crabmeat is a highly perishable commodity and is rendered unusable by the normal flora after 7 to 12 days of refrigeration. However, pasteurization kills most of the normal flora and extends the refrigerated shelf life to 6 months or more, well beyond the time required for toxin development at 40 F in crabmeat homogenates. Although the homogenates did become toxic at 40 F after 55 days or longer, inocula of 10' spores per tube or higher were required. Pasteurization reduced spore loads as high as 108 spores per 100 g to 6 or less spores per 100 g, and the meat remained nontoxic during 6 months of storage at 40 F. It is apparent from the limited survey of bottom mud samples reported that C. botulinum type E is commonly found in the environment from which blue crabs are harvested. It would follow that the organism is likely to be present on the crabs as they are brought into the processing plants. Though the spores would undoubtedly be killed by a recommended cooking of 250 F for 10 min (12), it is possible that the cooked crabs or picked meat could become contaminated by spores present in the plant. Preferably, crabmeat is pasteurized immediately after picking (12); however, regulations of the Maryland State Department of Health and Mental Hygiene (10) allow the meat to be refrigerated for up to 24 h prior to pasteurizing. Unless the meat is grossly mishandled, it appears highly unlikely that more than a few chance contaminants would be present at the time the meat is ready for pasteurization. Pasteurized meat of the blue crab has been available to the consumer for over 20 years and, to date, no case of botulism has been attributed to its consumption, nor has any commercially available pasteurized meat been found toxic. This excellent record coupled with the data in this report indicate that pasteurized meat of the blue crab is a safe product provided the prescribed procedures of cooking, handling, pasteurization, and storage are adhered to rigorously.

v3-fos

2018-04-03T00:17:03.922Z

1974-07-01T00:00:00.000Z

10802973

s2

Effect of Growth Rate and Nutrient Limitation on the Composition and Biomass Yield of Acinetobacter calcoaceticus Acinetobacter calcoaceticus was grown on ethanol in a chemostat as a model system for single-cell protein production. The substrate yield coefficient (Y8, grams of biomass/gram of ethanol), protein yield coefficient (Y1, grams of protein/gram of ethanol), and biomass composition were measured as a function of the specific growth rate. Nucleic acid, protein, Y,,, and Y, all increased at higher growth rates. Although protein content increased only 14% (from 53 to 67%), Y,, almost doubled over the same range of growth rates. The increase in Y. was due to the higher protein content of the biomass and to higher values of Y8. The higher values of Y8 were attributed to maintenance metabolism, and the value of the maintenance coefficient was found to be 0.11 g of ethanol per g of cell per h. When A. calcoaceticus was cultivated under a phosphorus limitation protein content, Y,, and Y, were lower than in carbon-limited cultures. It was concluded that a single-cell protein fermentation using A. calcoaceticus should be operated at a high growth rate under ethanol-limiting conditions in order to maximize both the protein content of the biomass and the amount of biomass and/or protein made from the substrate. was due to the higher protein content of the biomass and to higher values of Y8. The higher values of Y8 were attributed to maintenance metabolism, and the value of the maintenance coefficient was found to be 0.11 g of ethanol per g of cell per h. When A. calcoaceticus was cultivated under a phosphorus limitation protein content, Y,, and Y, were lower than in carbon-limited cultures. It was concluded that a single-cell protein fermentation using A. calcoaceticus should be operated at a high growth rate under ethanol-limiting conditions in order to maximize both the protein content of the biomass and the amount of biomass and/or protein made from the substrate. The composition of a microorganism is not invariant but is strongly dependent on its growth rate and environment. The chemostat is an ideal tool for studying changes in cell composition because both environment and growth rate can be controlled readily and accurately. By using chemostat cultures, it was found that there is a close correlation between specific growth rates (,u) and ribonucleic acid content of microorganisms (5,12,16,19,22). This variation is due primarily to the changes in ribosome content (6,10). Protein content also varies with growth rate, but unlike ribonucleic acid, the extent of variation usually is not great (10,21). The carbohydrate content usually does not change greatly when cells are grown at different rates under carbon-limiting conditions; however, chemostat cultures of nitrogenor sulfurlimited cells exhibit wide variations in carbohydrate content (10,21). The growth rate of a microorganism influences not only its composition but also the substrate yield coefficient (Y8; grams of biomass produced per gram of substrate consumed). Usually one of two types of relationships between ,g and Y8 are reported: (i) Y, increases as ,u increases, attaining a maximum value as the growth rate approaches /2max (13); or (ii) Y8 at-'Present address: Eli Lilly and Co., Dept. M539, Indianapolis, Ind. 46206. tains a maximum value at some intermediate growth rate (11). The first type of behavior has been attributed to the maintenance metabolism of the microorganism (13,14), which is a measure of the consumption of substrate by microorganisms for functions other than the production of new biomass. These functions include maintaining concentration gradients, providing energy for motility, resynthesizing unstable macromolecules, etc. The amount of substrate consumed for these purposes is a progressively smaller proportion of the total substrate consumption as the growth rate increases. Thus, more substrate goes to biomass production, causing Y, values to increase. This relationship can be expressed as: where (1/x) (ds/dt) is the specific rate of substrate consumption (grams of substrate consumed per gram of cell per hour), m is the maintenance coefficient (grams of substrate consumed for maintenance per gram of cell per hour), and Yg is the yield coefficient when m = 0 or whengu approaches infinity (14). By plotting (1/x) (ds/dt) versus A, m and Yg can be found as the intercept and the reciprocal of the slope, respectively. The chemostat, in addition to its usefulness for studying changes in biomass yield and composition, is the preferred method of cultivation for the production of single-cell protein. In developing this process, it is important to establish how biomass composition can be varied, since the composition is a determinant of the nutritional and toxicological properties of the product. The protein content of single-cell protein also influences its market value, and the cost of production is strongly dependent on the protein yield coefficient, Y,, (grams of protein produced/gram of substrate consumed). Thus, for single-cell protein production, it is desirable to increase Yp, Y,, and protein content, three factors that may vary with the growth rate of the microorganism or the nutrient limitation under which it is grown. MATERIALS AND METHODS Organism. The organism used was Acinetobacter calcoaceticus strain 4736. Taxonomic characteristics and procedures for maintaining stock cultures were described previously (1,2). Fermentation. A. calcoaceticus was cultivated in a mineral salt medium containing ethanol as a sole carbon source. The medium, temperature, and pH for chemostat cultivation were described previously (1). For ethanol-limited cultures, the entering medium contained 6 g of ethanol per liter. Similar medium, temperature, and pH were used for phosphoruslimited cultures, except that HPO4 was deleted from the medium and added as a dilute solution via a second stream. The rate of PO4 addition was gradually reduced until about 0.05% ethanol accumulated in the culture broth. At low growth rates, the chemostat was operated for longer periods of time before biomass yields and composition were determined. At least four changes of medium were made at all growth rates to insure steady-state conditions. The dissolved oxygen tension in the fermentor was continuously monitored by a galvanic electrode and was maintained at greater than 20% of air saturation by varying agitation and aeration rate. At each steady state the concentrations of ethanol, acetate, and acetaldehyde in the culture broth were measured (1,2). In ethanol-limited cultures, the steady-state concentration of these metabolites was less than 0.05 g/liter. In phosphate-limited cultures, the ethanol concentration in the broth was measured by quantitative gas chromatography (1,2) and subtracted from the amount added to obtain the amount of ethanol consumed. Analytical methods. (i) Cell mass. Cell mass was determined by dry weight measurements. A portion (5.0 ml) of the cell suspension from the steady-state chemostat was transferred into a tared centrifuge tube. The biomass was sedimented by centrifugation at 12,000 x g for 15 min, and the supernatant fluid was discarded. The pellet was-resuspended in 5.0 ml 0.1 N HCl, sedimented, and washed with distilled water. The washed pellet was dried at 105 C, cooled in a desiccator over CaCl,, and weighed. The amount of precipitated salts in carbon-limited cultures was about 0.3 mg/ml, but there was very little precipitation in phosphorus-limited cultures. The HCl wash removed precipitated salts that sedimented with the biomass, compensating for the differences in the two types of medium. Control experiments indicated that the HCl and distilled water washes did not cause appreciable cell lysis. The dry weight measurements were performed in quadruplicate, and the averages are reported. (ii) Carbohydrate. For total carbohydrate measurements, 0.5 ml of the biomass suspension was added to 5.0 ml of anthrone reagent (7). The mixture was placed in a boiling-water bath for 10 min and cooled, and the absorption was read at 620 nm in a Gilford 2400 spectrophotometer (Gilford Instrument Laboratories, Oberlin, Ohio). A standard curve was prepared by using glucose, and the determinations were preformed in triplicate. (iii) Lipid. A cell suspension (1.0 liter) was removed from the chemostat, centrifuged, and resuspended in 200 ml of cold 1.0 N HCl. This suspension was incubated for 1 h to remove the low-molecularweight (cold acid-soluble) components of the biomass. The supematant fluid was discarded, and the pellet, containing high-molecular-weight components, was resuspended in 200 ml of 10 -I N HCl. A portion of this suspension was retained for protein and nucleic acid analyses, and the remainder (100 ml) was used to determine lipid content. For lipid analysis, the biomass was extracted first with 30 ml of acetone at room temperature. The material remaining after the acetone extraction was extracted with a mixture of 15 ml of ethanol and 5 ml of ether at 100 C for 5 min. The solvents were recovered by centrifugation, and the remaining biomass was refluxed for 1 h with a mixture of 5 ml of methanol and 15 ml of chloroform. The solvent extracts were combined, filtered, dried, and weighed. (iv) Nucleic acids. The biomass pellet remaining after removal of the cold acid-soluble (low-molecularweight) fraction was used for protein and nucleic acid analyses. The pellet from 10.0 ml of suspension was resuspended in 0.5 N perchloric acid and incubated at 90 C for 45 min. The suspension was cooled and the supernatant fluid was recovered after centrifugation. The supernatant was adjusted to pH 7.0 with KOH and the absorbance at 260 nm was determined in a Gilford spectrophotometer. An extinction coefficient of 0.0315 cm2/4g was used to convert absorbance to nucleic acid concentration (8). (v) Protein. The cell pellet remaining after the perchloric acid-catalyzed hydrolysis of nucleic acid was used for the protein analysis. The pellet was resuspended in 1.0 N KOH and incubated at 90 C for 45 min. The reaction mixture was cooled and the supernatant fluid was used for a biuret protein determination (18). A standard curve was prepared with bovine serum albumin. Protein content also was expressed as (%N x 6.25) -% nucleic acid. Total nitrogen was determined by a carbon, hydrogen, nitrogen analysis (Perkin-Elmer 240 CHN analyzer, Perkin Elmer Corp., Norwalk, RESULTS A. calcoaceticus was cultivated in a chemostat under ethanol-limiting conditions. A steady state was obtained at various growth rates, and the substrate yield coefficient, carbohydrate, lipid, nucleic acid, and protein content were measured. Substrate yield coefficient. YB of A. calcoaceticus was strongly dependent on the growth rate, particularly at low specific growth rates. The specific rate of substrate consumption, (-) (d) = y, was calculated at each growth rate and plotted as a function of t (Fig. 1). A straight line was obtained that intersected the ordinate at a positive value, indicating that the variation of Y8 with At was due to maintenance metabolism. The values of m and Yg calculated from Fig. 1 of the biomass remained essentially constant when the growth rate was varied (Fig. 2), whereas nucleic acid varied from about 7.5 to 12% (Fig. 3). There were no large differences in protein content when protein was measured by the biuret assay; however, the results obtained were highly variable and these variations would obfuscate all but large changes. In addition, when protein was determined by an amino acid analysis, it was found that the protein content was Downloaded from much higher than that indicated by biuret assay. Closer agreement with the amino acid analysis and more consistent results were obtained by determining protein content from the nitrogen and nucleic acid content of the biomass (see Materials and Methods). The resultant data suggested that the protein content of A. calcoaceticus increased from about 53 to 67% of the biomass as the growth rate was increased from 0.1/h to 0.53/h (Fig. 2). Although the protein content of the microorganism varied by only 14%, the protein yield n .6- coefficent (Yr) almost doubled, increasing from 0.24 to 0.43 over the same range of growth rates (Fig. 4). This large variation was due to the additive effects of the higher values of Y., and the higher protein content of the microorganism. Phosphorus limitation. A. calcoaceticus was cultivated in a chemostat on ethanol under phosphorus-limiting conditions. A steady state was achieved at a specific growth rate of 0.5/h, and analyses were made of the biomass composition and the ethanol and phosphate yield coefficients. The steady state was maintained for 3 days, and daily measurements were made. Y8 was significantly lower under phosphoruslimiting conditions than under an ethanol limitation (Table 1). These yield coefficients were corrected for unused ethanol in the culture broths. The protein content appeared to be somewhat lower, whereas the total nucleic acid content was slightly higher (11.8 versus 14.2%) than in ethanol-limited cultures growing at a similar rate ( Table 1). As a result of the declines in protein content and Y,, Y,, was about onethird less in phosphorus-limited cultures. DISCUSSION Our data suggest that the protein content of A. calcoaceticus in an ethanol-limited chemostat increases when the growth rate increases. In contrast, others have reported that the protein content of microorganisms remains relatively constant or decreases when the growth (3,4,9,21). The protein content, as we measured it, was based on the total nitrogen content of the biomass. As a result, these protein values may be higher than the true protein content because of the presence of non-protein nitrogen other than nucleic acids (e.g., n-acetylglucosamine and muramic acid in cell walls). Nitrogen-based protein analyses also are dependent on the somewhat arbitrary extinction coefficient used for the total nucleic acid estimation. These sources of error influence primarily the magnitude of the protein content, but they should not hinder the determination of relative differences in protein values unless large changes in non-protein or non-nucleic acid nitrogen content occur. The identity of proteins responsible for the apparent increase in protein content of A. calcoaceticus at higher growth rates is not known. At least part of the increase may reflect the increase in enzyme level that presumably must occur to sustain high specific growth rates. Also, part of the increase may be due to ribosomal protein. Schaechter et al. (17) and Ecker and Schaechter (6) reported that the rate of protein synthesis per ribosome is independent of growth rate. Thus, larger numbers of ribosomes are needed to support higher growth rates. Although Sykes and Young (20) have shown that the rate of protein synthesis per ribosome increases with increases in growth rate, the rate of synthesis per ribosome was constant at growth rates above g = 0.5/h. Of particular significance to single-cell protein production is the variation of Y, with growth rate. This variation was due primarily to maintenance metabolism. At higher growth rates, the amount of substrate diverted to maintenance becomes a progressively smaller proportion of the total amount of substrate consumed. As a result, Y, increases at a greater rate than Y8 because both protein content and Y, are higher at higher growth rates. Earlier reports indicated that the protein content of a microorganism may be higher under a phosphorus limitation than under a carbon limitation (15,21). A similar increase in a single-cell protein fermentation would have a beneficial impact on process economics if concomitant decreases in Y, and Y, did not occur. The present study indicates that A. calcoaceticus contained slightly less protein in phosphorus-limited cultures, and this decrease was accompanied by substantial declines in Y, and Y,. In conclusion, a single-cell protein fermenta-tion using A. calcoaceticus should be operated under ethanol-limiting conditions at high growth rates in order to maximize the protein content of the product and the amount of protein and/or biomass produced from ethanol.

v3-fos

2020-12-10T09:04:12.211Z

1974-03-01T00:00:00.000Z

237232599

s2

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH4)2SO4; however, 1% (NH4)2SO4 favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH4)2SO4. They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared. (NH.)2S04 favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH4)2SO4. They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared. Aflatoxins are secondary metabolites produced primarily by some strains of Aspergillus flavus and Aspergillus parasiticus. Because of their pronounced toxicity and extreme carcinogenicity in many animal species, aflatoxins have been, and continue to be, extensively investigated (13,23). Results of limiteid tests (6,7,16) have revealed a close relationship between biosynthesis of lipid and aflatoxin. However, it has not been demonstrated that this relationship prevails under all cultural conditions. Experiments in our laboratory showed that the pattern for synthesis of total lipids and aflatoxin was similar when A. parasiticus grew with different degrees of aeration (C. N. Shih and E. H. Marth, Biochim. Biophys. Acta, accepted for publication). Detroy and Hesseltine (6) suggested that synthesis of aflatoxin may be controlled through changes in the environment that can direct precursors into lipid rather than toxin. This study was designed to define more completely the relationship between synthesis of lipid and aflatoxin by A. parasiticus. To accomplish this, the mold was grown in a synthetic medium so that variability in natural substrates was eliminated. Then the concentra-tion of glucose and nitrogen in the medium and incubation temperature were varied. These factors influence synthesis of lipids by some other molds (4,9,15), but their importance for synthesis of fat by toxigenic aspergilli has not been defined nor has it been established that the similarity between synthesis of lipid and aflatoxin prevails over a variety of cultural conditions. Results in this communication provide some of the missing information. MATERIALS AND METHODS Organism. A. parasiticus NRRL 2999, a toxigenic strain obtained from Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., was used throughout this investigation. Stock cultures were maintained at 5 C on slants of mycological agar (Difco). Preparation of spore suspension. The mold was grown on slants of mycological agar for 7 days at 28 C. Spores were harvested by adding sterile distilled water and a drop of Leconal wetting agent (Laboratory Equipment Co., St. Joseph, Mich.) to the slants. Each spore suspension was adjusted to an optical density value of 0.5 at 550 nm (Spectronic 20, Bausch and Lomb, Rochester, N.Y.) before it was used as inoculum. Media. A synthetic growth medium was developed 452 on the basis of earlier reports (5,11,14). The medium contained (per liter): 50 g of glucose, 6 g of (NHI)2SO4, 5 g of KH2PO4, 6.4 g of K2HPO4, 0.5 g of MgSO4 7H20, 2 g of glycine, 2 g of glutamic acid, 10 mg of FeSO4 7H20, 5 mg of ZnSO4 7H20, and 1 mg of MnSO4 H20. The finished medium was prepared by aseptically adding a sterile glucose solution to the sterile glucose-free salts solution when both were cooled. The pH of the medium was 6.4 to 6.5. This medium was used to prepare the inoculum and to study how temperature during growth affected lipid and toxin production. The medium used to study the effect of initial glucose concentration on formation of total lipid and toxin was prepared by eliminating glucose, glycine, and glutamic acid from the substrate just described and by adding various amounts of glucose, 0.5, 1, 5, 10, 20, 30, and 50% (wt/vol), to the solution. When various concentrations of nitrogen were desired, all nitrogen sources [glycine, glutamic acid, and (NH4)2SO4] were eliminated from the growth medium and different amounts of (NH4)2SO4, 0.05, 0.1, 0.5, 1, 3, 5, and 10% (wt/vol), were added to the nitrogen-free solution. Cultural conditions. In experiments to determine how the initial glucose and nitrogen concentrations affected biosynthesis of lipid and toxin, 2 ml of the spore suspension (7 x 106 to 8 x 106 spores/ml) were incubated quiescentiy with 100 ml of medium in a 500-ml Erlenmeyer flask at 28 C for 6 days. To study the effect of various temperatures on production of lipid and toxin, 2 ml of the spore suspension was incubated quiescently with 100 ml of the synthetic growth medium in a 500-ml Erlenmeyer flask at 25 C for 2 days. After this initial incubation, some flasks were allowed to remain at 25 C, whereas others were moved to storage at 15, 35, or 45 C. Molds at various temperatures were then incubated quiescently for up to 7 days. This procedure eliminated the problem of delayed spore germination at some of the temperatures. Samples were analyzed for toxin, lipid, glucose concentration, and mycelial dry weight after 3, 5, and 7 days of incubation. All experiments were done in duplicate and results are reported as average values. Glucose determination. Glucose was measured by the anthrone test (21).: Determination of mycelial dry weight. Samples of the culture were filtered using vacuum and four layers of cheesecloth on a Buchner funnel. After washing three times with cold distilled water, the mycelium was dried at 50 C for 24 h and weighed. Aflatoxin determination. A 20-ml amount of filtrate was extracted in a separatory funnel with 40 ml of chloroform, and this procedure was repeated three times. Aflatoxin in the mycelium was extracted by blending it with 50 ml of chloroform, 100 ml of methanol, and 40 ml of water in a Waring blender for a few minutes. Then 50 ml of chloroform and 50 ml of water were added to the mixture and it was blended again. After filtration, chloroform was removed from the mixture and the remaining methanol-water fraction was then extracted twice with chloroform (19). The combined chloroform extracts from either broth or mycelium were evaporated separately in a flash evaporator in preparation for aflatoxin analysis. Aflatoxins were separated on thin-layer chromatographic plates which were developed with chloroform-methanol-water (98:1:1, vol/vol/vol). The concentration of aflatoxins was measured using procedures described by Shih and Marth (18). Data in the figures represent the total aflatoxin (B., B2, G1, and G2) present in the broth and mycelium. Determination of total lipid. A portion of the chloroform extract was used to determine total lipid produced by the mold (1). The chloroform extract was dried by filtration over Na2SO4, and then chloroform was removed by flash evaporation. After chloroform was removed, lipid was transferred to a tared 50-ml Erlenmeyer flask with 4 to 5 ml of chloroform, which was then removed under a stream of nitrogen. The residue in the flask was dried over calcium chloride anhydride in a vacuum desiccator and weighed. RESULTS Initial concentration of glucose and synthesis of lipid and toxin. Data in Fig. 1 show that both the highest total lipid and the highest total aflatoxin concentrations were obtained with 30% glucose, although dry weight of the mold was maximal when the medium contained 10% glucose and it was nearly similar when the medium contained 5 or 20% glucose. Increases in initial concentrations of glucose up to 30% regularly resulted in greater amounts of lipid and aflatoxin produced by the mold. However, when the glucose concentration was increased from 30 to 50%, synthesis of both lipid and toxin was diminished and mycelial dry weight was reduced. Residual glucose in the medium increased markedly as the initial concentration was raised beyond 5%. Hence, the efficiency of glucose utilization by the mold decreased as the concen- tration was increased. The mold did not produce appreciably more acid when the medium contained more than 5% glucose. Initial concentration of nitrogen and synthesis of lipid and toxin. The influence of several concentrations of (NH,)2SO on biosynthesis of lipid and toxin is shown in Fig. 2. Maximal mycelial dry weight occurred when the medium contained 3% (NH,)2SO,. However, 1% (NH,)2SO4 favored maximum accumulation of total lipid and toxin. The results indicate that concentrations of nitrogen somewhat suboptimal for maximal growth (greater carbon-to-nitrogen ratio) enhanced lipid synthesis and production of aflatoxin. Presence of 5% or less (NH4)2SO4 permitted use of all glucose (5%) in the medium during the incubation of 6 days. Nearly 50% of the glucose remained unused when 10% (NH4)2SO0 was in the medium. Acid production by A. parasiticus was decreased when the medium contained more than 1% (NH,)2SO4. This coincided with the decrease in the amount of lipid and toxin that was produced. Incubation temperature and synthesis of lipid and toxin. Data in Fig. 3 indicate that maximal mycelial dry weight was produced at 35 C. However, maximal concentrations of total lipid appeared at 25 and 35 C in 2 to 5 days, and maximal concentration of aflatoxin appeared at 25 C in 5 days. Both lipid and aflatoxin were produced when A. parasiticus grew at 15 and 25 C (Fig. 3). At higher temperatures, 35 and 45 C, the mold continued to form lipid, but produced only negligible amounts of toxin. Results of these experiments demonstrate that a temperature which was somewhat suboptimal for growth of A. parasiticus permitted maximal production of both lipid and toxin. Furthermore, biosynthesis of aflatoxin by the mold was essentially completely suppressed at or above 35 C, whereas production of total lipid prevailed under these conditions. DISCUSSION Results from this study define some of the conditions under which synthesis of lipid and aflatoxin have a similar or dissimilar pattern. The close relationship between biosynthesis of lipid and aflatoxin also has been suggested in limited studies by other investigators (6,7,16). However, they found that certain cultural conditions supported good production of aflatoxin but were unfavorable for synthesis of lipid. The reverse also was true. These observations led to the general conclusion that formation of more aflatoxin or more lipid can be controlled by manipulating the environment. Results of our investigation lead to another conclusion-manipulating the environment does not always favor production of one at the expense of the other. We noted a similar pattern for synthesis of lipid and aflatoxin at all concentrations of glucose and (NH,)2SO4 in the medium and when incubation was at 15 and 25 C. A dissimilarity was noted only when the mold grew at 35 and 45 C; production of lipid continued unhindered but formation of aflatoxin was suppressed. Maximal formation of lipid and aflatoxin by A. parasiticus was favored by a high concentration of glucose (30%) that did not support formation of maximal mycelial dry weight. A somewhat suboptimal (for maximal growth) concentration of ammonium sulfate (1%) yielded maximal production of lipid and aflatoxin. These conditions also were found (4,10,15,24). It was reported earlier that an increase in the carbohydrate concentration of the medium resulted in the synthesis of more total lipid by other molds (4,12,15,24). Lockwood et al. (12) obtained most total lipid when Penicillium javanicum grew in a medium with 30% glucose. A high concentration of carbohydrate is believed necessary to supply the carbon and energy needed for maximal synthesis of lipid (4,9,15). Since our results show that the highest concentration of aflatoxin appeared in the medium that also supported maximal synthesis of total lipid, a similar explanation may apply for aflatoxin synthesis, at least when proper environmental conditions prevail. That a high concentration (20%) of sucrose supports maximal production of aflatoxin has been reported (5). It has also been shown that an abundant supply of glucose in the medium caused a marked impairment of respiratory capability and concurrent development of an anaerobic mode of metabolism by many microorganisms (22). In other studies (20; Shih and Marth, Biochim. Biophys. Acta, accepted for publication), we showed that conditions bordering on anaerobiosis favored synthesis of aflatoxin. That observation also may help to explain why the maximal yield of aflatoxin appeared in a medium with a high concentration of glucose since the mold probably developed a less aerobic mode of metabolism in response to the excess glucose. According to Bu'Lock (2), the concentration of nitrogen in the medium is critical for synthesis of secondary metabolites. We observed that a nitrogen concentration somewhat suboptimal for maximal growth produced maximal yields of lipid and aflatoxin. This also was found true for other molds when conditions for maximal yield of lipid alone were studied (4, 10, 15). Since a similar C: N ratio is optimal both for synthesis of aflatoxin and lipids, aside from the similar precursor acetate (6, 7), perhaps another condition(s) is common to the synthesis of both products. Formation of lipid and aflatoxin varied with the temperature of growth used after an initial 2-day incubation at 25 C allowed conidia to germinate. Maximal synthesis of aflatoxin occurred at 25 C and of lipid at 25 and 35 C, whereas maximal mycelial dry weight was produced by A. parasiticus at 35 C. The observations made in these trials verify those of Schindler et al. (17), who studied only aflatoxin production and reported that highest yields were obtained at 24 C, whereas maximum mold growth occurred at 29 or 35 C, depending on the particular isolate. At each of the lower temperatures, 15 or 25 C, we observed that formation of both products followed a somewhat similar pattern. At higher temperatures, 35 or 45 C, lipid was produced preferentially and only small amounts of aflatoxin appeared. Previous studies by Ciegler et al. (3) and by Diener and Davis (8) also showed that aflatoxin formation was limited by high temperatures. Our observation and those of others suggest that a key enzyme responsible for aflatoxin synthesis was inactive above 35 C, and thus it is possible to regulate aflatoxin production through control of temperature. Our results demonstrate that similarities exist between synthesis of lipid and aflatoxin under a series of different conditions. This was not observed by other workers. Our data also indicate that certain conditions (e.g., incubation at 35 or 45 C) favor lipid synthesis and suppress production of aflatoxin. These observations suggest that formation of both products shares some similar biosynthetic steps. Perhaps a key enzyme responsible for synthesis of either lipid or aflatoxin is regulated by certain cultural conditions and hence the condition(s) determines the amount of the final products that will appear. Data from another study in our laboratory (C. N. Shih and E. H. Marth, unpublished data) support the idea that some similar biosynthetic steps are involved in formation of both lipid and aflatoxin. These data suggested that the same pathway is used initially in the biosynthesis of lipid and aflatoxin through condensation of acetate and malonate units and that formation of polyacetate precedes production of aflatoxin.

v3-fos

2020-12-10T09:04:22.666Z

1974-09-01T00:00:00.000Z

237233349

s2

Development of a Selective Enterococcus Medium Based on Manganese Ion Deficiency, Sodium Azide, and Alkaline pH Rogosa broth, without its salt supplement and dissolved in deionized water, was adapted for the selective isolation and enumeration of enterococci. This medium supported good growth of enterococci, but it suppressed growth of other lactic acid bacteria. The sensitivity and specificity of the medium were tested after addition of various increasing concentrations of NaN3 against known strains of enterococci and other bacteria. Many strains of Streptococcus faecium showed low azide tolerance; optimal growth was obtained at a concentration of 0.01% NaN3, which totally or partially inhibited unrelated species of lactic acid bacteria. The selectivity of the medium was further increased by pH adjustment to 9.6. Carbonate and Tween 80 were added to overcome partial inhibition of enterococcal growth by the new combination of selective conditions. The final medium was evaluated in agar form in isolations from human and animal feces, polluted water, meat, and dairy products. Counts were obtained after 16 to 17 h of incubation at 37 C. The isolates satisfactorily conformed to the group characteristics of enterococci. Depletion of available Mn2+ levels in nutrient media results in a significant reduction of growth by lactobacilli (7) and pediococci (6), but has no effect on the growth of enterococci (6). Since the former groups of bacteria frequently occur in nature together with enterococci, the utilization of Mn2+ deficiency might facilitate the isolation of enterococci from mixed microfloras. A selective medium utilizing this premise was developed in the present study. The sensitivity and specificity of the medium was tested against known strains of bacteria and in isolations from various natural sources. (This investigation was presented in part at the 73rd Annual Meeting of the American Society for Microbiology, Miami Beach, Florida, 6-11 May, 1973.) MATERIALS AND METHODS Cultures. Authenticated strains of enterococci were used for the initial evaluation of the medium. They comprised Streptococcus faecalis, American Type Culture Collection (ATCC) 11700, 14428, and strain 19-1, obtained from W. H. Seeley, Cornell University; S. faecium typical strains, ATCC 6057, and 13 other strains from the culture collection maintained in this laboratory. The latter strains had been previously isolated from dairy products and identified according to Deibel (3,4); S. faecium atypical strains, FCMA-2 (var. casseliflavus) and FCMA-11, were obtained from J. 0. Mundt, Univer-sity of Tennessee, and T-15 (a Langston strain isolated from grass silage) was received from H. W. Seeley. In addition, we included three strains of S. bovis, SS-752, 963, and 964, and three strains of S. equinus, SS-945, 946, and 947, received from R. R. Facklam, Center for Disease Control, Atlanta, Ga. Other lactic acid bacteria, not related to fecal streptococci, included: S. lactis ATCC 7962, S. cremoris ATCC 9625, S. diacetilactis DRC-1, and two group N strains received from Rebecca Lancefield, Rockefeller University, New York; Leuconostoc mesenteroides, two strains obtained from J. 0. Mundt, L. dextranicum Ld688 and L. citrovorum 91404, obtained from W. E. Sandine, Oregon State University, and L. citrovorum Al, obtained from T. W. Keenan, Purdue University; Pediococcus cerevisiae ATCC 8081, 8042, 10981 and strain no. 25, obtained from H. W. Seeley; Lactobacillus bulgaricus G5 and L. jugurti G2, isolated from Greek yogurt by C. J. Efthymiou; and one strain each of L. casei and L. brevis, obtained from the Midwestern Culture Service, Terre Haute, Ind. Five strains of different gram-negative and gram-positive organisms with cytochromeand catalase-dependent metabolism (see Table 3) were also included to test the selective capacity of the NaN,-containing medium. Culture media. As basal medium we used a modified Rogosa broth (6); the salt supplement of the complete medium was deleted; all ingredients were dissolved in deionized, all-glass-distilled water. NaNs was added at different, increasing amounts: 0.01 0.015, 0.02, 0.025, 0.03, and 0.05%. The resultant media were evaluated against our test cultures. The concentration adopted for the final medium was 0.01% NaNs. Various initial pH values were also examined, and we finally selected pH 9.6 as the most suitable; in conjunction with this modification, 5.3 g of Na2CO, per liter was added. The final selective medium of this study (Table 1) was prepared in the following manner: Trypticase, yeast extract, tryptose, ammonium citrate, Tween 80, sodium acetate, and glucose were dissolved in 150 ml of water; the K2HPO4 salt was dissolved in 50 ml of water; the carbonate was dissolved in 50 ml of water and was autoclaved in a screw-capped bottle with its cap tightened. All three solutions, autoclaved for 15 min at 15 lb/in2 (121 C), were mixed aseptically at room temperature, and the pH was adjusted to 9.6 with sterile 2 N NaOH. Five milliliters of 2% Seitz-filtered NaN, was added. The temperature of the medium was then raised to 70 C in a water bath, and a sterile solution of agar (15 g dissolved in 740 ml of water) was added and mixed well. After mixing, the pH was rechecked and, if necessary, readjusted. Avoiding direct exposure to sunlight, the medium was poured into plates, allowed to solidify, and stored in a refrigerator. The medium was also tested as broth (agar omitted). All dissolved ingredients making up the broth were mixed aseptically at room temperature. For application, a sterile membrane filter (pore size 0.22 gm) was aseptically placed in a sterile filter holder. Ten milliliters of each cheese solution tested was introduced into the funnel and vacuum was applied. The funnel wall was rinsed with 50 ml of sterile phosphate buffer (pH 7.2). After filtration, the filter was aseptically transferred to a plastic petri dish containing an absorbent pad already saturated with about 2 ml of broth, and incubated. Enterococcus counts of various natural samples were determined on this medium. Total viable counts of the same samples were obtained on standard methods agar (BBL). Culture methods. The selective medium was tested in both liquid and agar forms. Inocula of the test organisms were prepared by culturing each strain in basal broth. One drop of a 24-h culture was added from a sterile Pasteur pipette to each tube containing 8 ml of medium. In those cases where gram-negative organisms were tested (Table 3), the inocula were alternatively prepared by diluting the 24-h cultures 1:100 in phosphate-buffered saline (pH 7.0). For the Water, all-glass-distilled and deionized, to 1,000 ml (pH adjusted to 9.6) inoculation of agar plates, one drop of culture was spread in a standard three-way streak on the surface of the medium; the plates were incubated aerobically. All test media were incubated at the optimal temperature for each species tested. All enterococcus cultures and the isolation plates inoculated with natural specimens were incubated at 37 C, except for some tests, which, as indicated, were incubated at 45 C. Isolation of enterococci from natural sources. Fecal samples from cattle, sheep, swine, chickens, and ducks were obtained at the farm of the State University of New York, Farmingdale, N.Y. Human and dog fecal specimens were also used. Freshly voided samples were taken in sterile bottles and refrigerated until analysis. Five-gram samples were weighed, transferred aseptically into 100 ml of sterile phosphate-buffered saline (pH 7.0), dispersed for 45 s in a Waring blender, and then serially diluted. Portions of 0.1 ml were spread on the surface of agar plates with a bent glass rod. Water samples were taken from rivers, creeks, and nearshore points in the environs of New York City and Long Island. Volumes of 50 ml were centrifuged at high speed, the supernatants were siphoned off, and the sediments were taken up in 2 ml of sterile distilled water. Portions were deposited on agar plates and spread as described. Food samples included meat sausages and three cheese varieties purchased at a local New York market. The preparation of these samples for analysis followed established procedures (9,13). After enumeration at 24 and 48 h, colonies from all specimens cultured on the selective agar were picked and seeded in basal broth. The isolates were examined for conformity to the characteristics of enterococci (Sherman's criteria). They were also checked for catalase production and for group D antigen. The latter test was carried out microscopically by using a fluorescein-conjugated, group D-specific antiserum (Sylvana). RESULTS The addition of increasing concentrations of NaN3 to the nutrient base afforded an initial evaluation of sensitivity. Table 2 presents the effect of such additions of NaN3, on the growth of 20 enterococci and 18 lactic acid bacteria. At a NaN3 level of 0.02%, S. faecium grew weakly or not at all; when the azide level was decreased to 0.015%, most typical S. faecium strains grew well, and at 0.01% azide, even atypical variants isolated from plants grew well. S. faecalis, as expected, exhibited greater tolerance toward NaN,, as all strains grew at concentrations 0.02 to 0.05%. Certain of the tested lactobacilli, pediococci, and lactic streptococci showed equal or greater resistance than S. faecium to NaN3. The basal medium, supplemented with 0.01% NaN,, provided a maximum limit for satisfactory growth of the typical and atypical strains of S. faecium ( Fig. 1 and 2), but 0.01% NaN3 was insufficient to completely inhibit other lactic cocci and bacilli. Incubation at 45 C, tried as a supplementary selective factor, proved unsatisfactory, since some of our enterococci failed to grow within 24 h and, in some cases, even 48 h. All enterococci grew well in the basal medium adjusted to pH 9.6, whether in the presence or absence of 0.01% NaN, ( Table 3). The high alkaline pH inhibited growth in all six strains of non-enterococcal group D streptococci. To ascertain the alkaline growth limits of these strains, we varied the pH of the medium and adjusted it to lower values. We found that they could grow well at pH 8.9. The 10 strains of lactic cocci grew slowly in the alkaline, NaN,free medium within a 72-h period; only half of these strains could show some growth in the alkaline medium supplemented with 0.01% NaN. The pediococci and lactobacilli were unable to produce visible growth even after 72 h, either in the alkaline, NaN,,-free medium, or in the alkaline, 0.01% NaN,-containing medium. The five cytochromeand catalase-containing, gram-positive or gram-negative strains demonstrated a slight retardation of growth in the alkaline, NaN,-free medium, but they showed a marked retardation of growth and no visible growth before 24 h of incubation in the alkaline medium containing 0.01% NaN. This partial inhibition became more distinct when we used diluted (1:100) inocula. On the alkaline agar medium (pH 9.6 plus 0.01% NaN3), the enterococcus counts from cured meat, cheese, water, and feces (Table 4) varied between less than one enterococcus per ml of water to 20 x 108 per gram of sheep feces. The ratios of enterococci to the total count also varied; in the chicken feces, the enterococci were nearly as numerous as the total count, whereas in the dog feces the ratio was 1:60,000. Colonies of gram-negative bacteria that predominated in most of the fecal specimens, when grown on standard methods agar, were effectively suppressed by the selective medium ( Medium on the left contains 0% NaN, (control). 3). On the standard methods agar, the food samples yielded mostly small or pinpoint-size colonies, typical of gram-positive bacteria. On the selective medium, the colonies exhibited uniform morphology, whereas microscopically, they yielded gram-positive cocci. Upon prolonged incubation, i.e., 48 to 72 h, on some of the selective agar plates that were seeded with cheese samples, some hazy growth appeared around the well-developed enterococcus colonies (Fig. 4). Microscopic examinations revealed pleomorphic gram-positive bacilli. The medium used in these experiments was prepared with common-grade bacteriological agar. When purified, demineralized agar (Oxoid, Ion Agar no. 2) was substituted, the secondary growth was not observed. The observation indi-cates that inorganic ion depletion enhances the selectivity of the enterococcus medium. The round surface colonies obtained on the selective medium reached an average diameter of 1 to 2 mm after 16 to 17 h of incubation at 37 C. One hundred ninety-four colonies isolated at random from the 14 analyzed specimens (Table 4) conformed substantially to the Sherman criteria for enterococci; few possessed catalase, and they reacted well with group D, fluorescein-labeled antiserum. Among 151 isolates from the fecal and water samples, there were six catalase-positive strains. Such strains were not encountered at all among the isolates from the fermented meat and dairy products. Thirtyeight out of 42 strains, tested with the group D antiserum, produced reactions of a 2+ or higher intensity, whereas four showed a weaker reaction or no reaction at all. A few isolates showed nonconformity with one or another of the Sher-, man characteristics. Lack of agreement in more than one of the Sherman characteristics was observed in few of these variants. DISCUSSION About 20 years ago, Reinhold et al. (8) developed a selective medium containing 20 g of sodium citrate per liter and 0.01% NaN3. At this concentration, the NaN, had generally no adverse effect upon the numbers of enterococcus colonies that developed on the agar plates, although a slight inhibition of some unidentified enterococci was noticed. In higher concentrations (0.015 to 0.025%) a decrease in colony size was noted. At 0.03% NaN3, both the size and number of colonies were markedly inhibited. According to Reinhold et al. the 0.01% level of NaN8 showed highest sensitivity, not only in terms of recovery rates, but also in readability of colonies on the agar plates. We adopted the 0.01% concentration of NaN8 as the desired level of chemical inhibitor for our basal medium because of its least inhibitory effect on the growth and colony development of diverse forms of enterococci (Table 2, Fig. 1 and 2). We included a moderate concentration (2 g/liter) of citrate; as an energy source, we primarily used glucose. For a suppression of lactobacilli and physiologically related bacteria, we depended in part on Mn2+ depletion (5, 7). Adjustment of pH to 9.5 to 9.6 was utilized originally by Sherman and Stark (10) as a tolerance test for the identification of S. faecalis, and later by Smith and Sherman (11)and others as a general differential characteristic of the entire group of enterococci. Certain forms of enterococci, however, grew poorly or not at all at this pH, especially after short incubation periods. Chesbro and Evans (2) studied the factors affecting the growth of enterococci in highly alkaline media. They found that the addition of carbonate and oleate (Tween 80) markedly stimulated the growth of enterococci in media with a pH higher than 9. When our basal medium was supplemented with carbonate, the high alkalinity of the modified medium (pH 9.6) did not interfere with the growth of any of the 20 strains tested (Table 3). It is noted that Burkwall and Hartman (1) found that the addition of (12) observed that 0.01% NaN, in blood agar was the critical concentration for prevention of spreading growth of Proteus sp. and coliform bacteria, since a 0.008% level of NaN5 allowed such growth to occur. The delayed growth of the cytochrome-possessing strains in our alkaline plus 0.01% NaN.-containing medium suggests that the nutrients of this medium may favor a certain degree of bacterial resistance. Raising the level of-NaN, to higher than 0.01% levels would probably eliminate the growth of these strains. Nevertheless, we did not attempt such an increase since we intended to maintain the established sensitivity for enterococci at the 0.01% level, and also because isolations from fecal samples (Fig. 3, Table 4) indicated an efficient, for all practical purposes, suppression of gram-negative bacilli. In the isolations of enterococci from various natural habitats (Table 4), the enterococcus counts were determined within 16 to 17 h of incubation. This early enumeration proved useful from the selection point of view. Bacteria unrelated to enterococci that were able to grow on the experimental medium slowly or marginally (Table 3, Fig. 4) did not interfere with the presumptive count and isolation of the enterococci. Partial identification of the isolated strains disclosed an overwhelming proportion of confirmed enterococci ( Table 4). The environmental stress, to which enterococci are subjected in nature and particularly in foods, requires optimal growth conditions during isolation. Our results indicate that such optimal conditions needed for cell resuscitation may be very important for biotypes of enterococci with low tolerance toward the selective agents used in the isolations. The initial evaluation of the medium indicates a favorable performance on this essential point. A comparison of the developed medium with several standard enterococcus selective media is the subject of another report (5).

v3-fos

2020-12-10T09:04:17.242Z

1974-04-01T00:00:00.000Z

237233082

s2

Preparation of Free Heat-Resistant Ascospores from Byssochlamys Asci When Byssochlamys is grown for production of ascospores, some of the asci break up into their constituent ascospores, whereas others do not. For heat resistance studies, it is desirable to prepare a uniform suspension of free ascospores. This was accomplished with the aid of a pressure cell, from which a suspension of asci under high pressure was released to atmospheric pressure through a small orifice. Spores so treated had about the same heat resistance as untreated spores. When Byssochlamys is grown for production of ascospores, some of the asci break up into their constituent ascospores, whereas others do not. For heat resistance studies, it is desirable to prepare a uniform suspension of free ascospores. This was accomplished with the aid of a pressure cell, from which a suspension of asci under high pressure was released to atmospheric pressure through a small orifice. Spores so treated had about the same heat resistance as untreated spores. Byssochlamys fulva and B. nivea are heatresistant fungi that cause spoilage in canned and bottled fruit juices and juice mixes and in some canned fruit. First noticed in England in the early 1930s, these fungi have since caused spoilage outbreaks in Europe, Africa, North America, and Australia. In the processing of fruits and juices, the heat treatment is kept mild to avoid flavor damage, and Byssochlamys can survive the heat treatment and grow in the finished product. It is believed to cause no health hazard; it is a matter of the esthetics (2). These fungi are ascomycetes with spherical, eight-spored asci. The ascospores are believed to be the heat-resistant stage in the life cycle (1). Electron micrographs show a thin, structureless membrane surrounding the ascospores in the ascus (7), but the light microscope usually shows only the ascospores in their typical arrangement without any surrounding structure. MATERIALS AND METHODS Production of asci. The asci were produced by growing B. fulva strain NRRL 3493 on a thin layer of unacidified Difco potato dextrose agar for about 30 days at 30 C. The mycelium was then scraped, and the resulting mixture of hyphae and asci was suspended in water, from which the hyphae settled out so that the asci could be decanted as previously described (3). In our experience, conidiospores are rarely if ever seen with the asci. In this treatment, some of the asci were broken and their ascospores were released. To obtain a uniform spore preparation, it was therefore necessary to break the intact asci into ascospores or to separate them from the free ascospores. Spore counts. To determine the relative numbers of asci and ascospores in a population, differential counts were made with a phase microscope at x600. Relative frequencies of damaged and undamaged spores were determined in the same way. Dormant ascospores (either within the ascus or solitary) are refractile when seen under the phase microscope (3). For heat resistance determinations, counts of viable asci and ascospores were made by plating on unacidified potato dextrose agar. These counts are not very precise, because Byssochlamys forms very thin, rapidly spreading colonies, which are difficult to see when small. As a result, we planned for about 30 colonies per plate and made several replications to partly compensate for the low numbers of colonies. Nevertheless, the precision is lower than in most plate counts of bacteria. Heat resistance. Heat resistance was determined by using a thermal death-time flask (4, 5). Grape juice was preheated to 86 C and continuously stirred in a heated, three-neck flask. It was inoculated at zero time, after which samples were withdrawn at intervals for plate counts. This method is suitable for heat resistance determinations at temperatures below 100 C. RESULTS AND DISCUSSION Preparation of free ascospores. Asci were subjected to several treatments (Table 1, treatments 1-4) in order to break them into ascospores. None were completely successful, although treatments 2 to 4 could be used if the asci and ascospores could be subsequently separated. It is also reported that blending in a microhomogenizer (Sorvall) broke only 10% of the asci (9). Sublethal heating with stirring in a thermal death-time flask at 86 C (the method described above for heat resistance) caused almost complete breaking of the asci into ascospores in 5 min. However, without stirring, the asci remained intact, and others have reported (9) that asci remained intact when heated. Evidently the heat breakage of asci occurs under special conditions but is not a general phenomenon. Furthermore, ascospores heated at this temperature were heat shocked or heat damaged, and some probably were killed. Separation of free ascospores from asci by filtration has been reported (6). To test this method, we sonicated a suspension of asci and passed it through a Teflon filter membrane (30-60 ,um pore size; Chemware 75-X) to remove debris and then through a 75-M membrane (10-20 jim pore size). This should have been just small enough to retain the asci while passing the ascospores. In fact, although a few ascospores came through the filter, it plugged up so quickly that the yield was extremely low. Saccharomyces asci have been broken into their constituent ascospores (8) by incubation in a solution of Pronase (Calbiochem, Los Angeles, Calif.) followed by passage through a pressure cell (Aminco French pressure cell). When Byssochlamys asci were suspended in water and passed through the pressure cell, 99% or more of the ascospores were liberated from the asci (Table 1, treatment 6). This result was obtained regardless of the presence of Pronase, which was therefore omitted from further trials. The liberated ascospores formed large aggregates that could not be dispersed by shaking. Addition of 1 or 0.1% Tween 80 to the suspension before the pressure cell treatment prevented this, except for a few small groups of two to several ascospores after some treatments. When the treated ascospores were examined by microscopy, it was noted that some showed evidence of damage. Usually these had surface defects. A few had lost their refractility, thus showing evidence of internal change. A trial of several pressures showed that apparent damage to the ascospores was much more frequent at high pressures (Table 2). However, the plate counts were about the same throughout the pressure range, suggesting that the visually observed damage was superficial. Nevertheless, to minimize the possibility of damage, the pressure range of 4,000 to 9,000 lb/in2 is recommended. Theoretically, plate counts of the free ascospores should be almost eight times as high as those of the untreated asci. Actually they are about four times as high. This is probably because some of the free ascospores are dormant and fail to grow in time to be counted on the plates. If germinating asci are observed by microscopy, it can be seen that the eight ascospores never germinate simultaneously. Thus, when they are separated and plated, some do not form colonies soon enough to be counted. For the data shown in Table 2, samples were subjected to a flow rate out of the pressure cell of about 3 ml/min. At 9,000 lb/in2, a flow rate of 20 ml/min had the same effect. At 3,800 lb/in2, the 20 ml/min rate may have been somewhat less effective. The flow rate was 3 ml/min elsewhere in these experiments, although a higher rate would probably be acceptable for treatment of large amounts of material. Heat resistance of free ascospores. To show that ascospores retain their heat resistance after this treatment, we compared treated and untreated spores. The free ascospores had about the same heat resistance as the asci from which they came, as shown by a thermal death-time plot for untreated asci and treated ascospores at 86 C (Fig. 1). Each line is the most probable (best least squares) fit to the corresponding points. That the lines have nearly the same slope shows that the treatment did not greatly change the heat resistance of the spores. As previously stated, plate counts on Byssochlamys lack precision, and this probably accounts for the scatter in the points.

v3-fos

2020-12-10T09:04:20.524Z

1974-09-01T00:00:00.000Z

237231542

s2

Improved Isolation and Differentiation of Enterococci in Cheese Further documentation of an enterococcus selective differential (ESD) medium was obtained in isolations from eight different cheeses. An improved differentiation of tetrazolium salt (2, 3, 5-triphenyl tetrazolium hydrochloride [TTC])-reducing strains of Streptococcus faecalis from TTC-nonreducing or TTC-faintly-reducing Streptococcus faecium was attained. The sensitivity of the medium was evaluated in comparison with that of KF streptococcal, Pfizer selective enterococcus (PSE), the medium of Reinbold, Swern, and Hussong (RSH), and the medium of Saraswat, Clark, and Reinbold (SCR). Selective counts, rate of colony formation, and ease of isolation and differentiation of colonies were examined. The specificity of the medium was also investigated. ESD supported the fastest rate of growth and the maximum size of colonies; counts in this medium were in most cases possible with 17 h of incubation, whereas the other media required 24 to 48 h. A presumptive identification of 1,077 isolates by four biochemical tests disclosed that SCR, RSH, and ESD selected high, comparable percentages of strains that approximated most closely the typical description of enterococci (66, 60.1, and 58%, respectively). Low percentages (21.1 and 30.7%) were yielded by KF and PSE. The utility of ESD for a rapid, presumptive identification of enterococci was confirmed by serological and biochemical testing of color TTC-differentiated colonies isolated from 18 cheeses. A selective medium suitable for the isolation of enterococci was previously developed by incorporation of a low sodium azide concentration (0.01%) and a high alkaline (pH 9.6) into a manganese-deficient nutrient base (5). In the initial evaluation of this medium, known pure strains of bacteria were used. In the present study, we extended the utility of the medium by including in its composition a tetrazolium salt indicator effecting presumptive speciation of the enterococcal isolates. We also tested the selective and differential capacities of the medium on various cheeses. Comparative isolations of enterococci were conducted by using several standard media. We made observations on the sensitivity of enterococcal recovery; selective counts, rate of colony formation, ease of isolation, and differentiation were considered. For an evaluation of specificity of the medium, we carried out a comparatively presumptive identification of cheese isolates. Finally, we examined the practical features of the medium in a few parallel applications of its solid and liquid form. MATERIALS AND METHODS Culture media. The medium tested in this study, designated enterococcus selective differential (ESD), was that which was described in a previous study (5), modified to include a tetrazolium salt indicator. After addition of sterile agar, the temperature of the medium was decreased to 50 C, and 5 ml of a 2% 2,3,5-triphenyl tetrazolium hydrochloride (TTC), sterilized previously in flowing steam for 30 min, was added. This medium was also tested as ESD broth (agar omitted). In this case, the sugar was autoclaved separately as a concentrated (10%) solution and its final concentration was reduced to 1% (the glucose level of Barnes TG (1) medium with which ESD was compared). The following agar media were used in addition to ESD: KF-streptococcal (BBL), Pfizer selective enterococcus (PSE), the medium of Reinhold, Swern, and Hussong (RSH; 14), and the medium of Saraswat, Clark, and Reinbold (SCR;15). Standard methods agar (SMA;BBL) was used as a nonselective control. Yeast extract (YE) agar was used as recovery and maintenance medium for all isolates from cheese. The composition of this medium was: yeast extract (20 g), K2HPO4 (2 g), MgSO4.7H2O (0.1 g), glucose (2 g), agar (15 g), and distilled water to 1,000 ml. The final pH was adjusted to 6.8. The same composition, but without agar, was used as YE broth for preparation of inocula in the presumptive identification of cheese isolates. TTC reduction in ESD media. The kinetics of TTC reduction by enteroccocci were studied comparatively in the standard TG medium of Barnes (1) and ESD broth. For this purpose, the same concentration of TTC (75 ug/ml) was added to both media. Each tube, containing 5 ml of TTC medium, was 417 inoculated with 3 drops of a 24-h culture. The reducing activity of known strains of enterococci, representing different physiological types, was examined visually every hour between 0 and 8 h, and at 24 h. Reduction was demonstrated by the appearance of magenta-colored triphenyl tetrazolium formazan. The reduced TTC was extracted with n-butanol. The optical density of the extracts was measured at 575 nm. Several concentrations of TTC were added to the selective ESD broth and were tested for a determination of the optimal concentration, i.e., the amount that was nontoxic and produced the best visible results. The same TTC concentrations were also incorporated in ESD agar plates which were then surface inoculated with the test organisms. At 0.01%, added to both liquid and solid media, no inhibition of cultures was noted, and the color of positive reactions was intense. Afterwards, this concentration was used routinely with both forms of ESD. Cheese samples analyzed. Eight different samples of cheese were used for the comparative isolation, enumeration, and presumptive identification of enterococci. An additional collection of 18 cheeses was used for the isolation, presumptive, and confirmed identification of isolates from the ESD medium only. All cheeses were obtained in retail packages from the local New York market. They represented cheese varieties differing as to country of origin, manufacturing procedures, species of animal that produced the milk manufactured into cheese, and environmental conditions prevailing at the time of manufacture and early ripening. All samples had been ripened for at least 60 days; some samples had been ripened for more than 6 months before they were marketed. Isolation and enumeration of enterococci. The handling, preparation, and analysis of the cheese samples were performed according to recommended methods (17). Serial, 10-fold dilutions were plated in SMA to determine the total viable count and in the selective media for comparative counting and isolation of enterococci. In the selective agar media, the size of inoculum was 0.1 ml; it was spread evenly on the surface of the agar using a bent glass rod. All cheese dilutions were inoculated in triplicate. The plates were incubated aerobically at 37 C. Counts were determined at 17, 24, and 48 h. Individual colonies were isolated by using the random sampling method of Harrison (9) after 48 h of incubation, and 30 to 60 colonies were picked per sample and medium. The counts were compared by the F test. For an assessment of sensitivity, the average size of 10 representative colonies, developed on the surface of each medium, was determined. The size or diameter of each colony was measured in 0.1-mm units on a Quanti Plate viewer (Kallestad Lab., Minneapolis, Minn.) apparatus utilizing dark-field lighting and a magnifying optical comparator, ordinarily used to measure immunoprecipitation patterns in agar gels. The relative sensitivity of the media was established by statistical analysis of the mean diameter of the enterococcus colonies by the Student-Newman-Keuls test. This is a test of multiple comparisons among means based on equal sample sizes (in this study, 10 colonies per plate), and it is derived from the principles of analysis of variance (16). Presumptive identification of enterococci. The cheese isolates obtained from the five selective media were inoculated in YE agar stabs. When growth was obtained, the cultures were placed in a refrigerator, where they were kept until further testing. For a presumptive identification of enterococci, the following five tests were performed: catalase activity, ability to grow on bile esculin medium (BEM), SF (Difco) medium, 0.1% methylene blue milk (MBM), and 6.5% NaCl broth. The latter four tests were performed and interpreted as a battery of tests. They were carried out according to the methods of Facklam and Moody (7). Catalase activity was tested in YE broth cultures. Inocula for all five tests were also grown in YE broth. The tests were adapted to utilize the multipoint inoculation system of Lighthart (12) and were read after 24 h of incubation at 37 C. The evaluation of relative specificity of the five media for enterococci was based on a statistical analysis (chi-square method) of the comparative identification data. Confirmed identification of enterococci. Colonies from 18 different samples of cheese, upon isolation from ESD agar, were transferred into serological tubes containing 2.5 ml of ESD broth. These cultures were incubated for 4 h at 37 C and then were examined for TTC reduction. The identification tests included the following. The morphology of isolates was examined by the gram stain. Presumptive verification was according to Sherman's criteria, the catalase reaction, and appearance on BEM (Difco). A screening for group D antigen was carried out by the Lancefield precipitation method in capillary tubes; for this test, a grouping antiserum was prepared in rabbits using the strain Lancefield D76 (S. faecalis var. zymogenes ATCC12958). Antigenic extracts for precipitation were produced from cells cultured in glucose Lemco broth according to Medrek and Barnes (13). Differentiation into species was determined in representative TTC-reducing and TTC-nonreducing strains, by testing for tolerance to potassium tellurite, fermentation of sorbitol, mannitol, arabinose, melibiose and raffinose, determination of folic acid requirement, substrate utilization of pyruvate, malate, and serine, and ability to grow in broth at 50 C. These tests were essentially performed according to Deibel (2,3). RESULTS TIC reduction in ESD. A summary of results is presented in Table 1. In the TG medium, the differentiation of typical S. faecalis from S. faecium by TTC reduction required 24 h of incubation. Intermediate enterococci, such as the epiphytic strains T-15, FMA 2, and FMA-11 (5), developed a positive reaction. These atypical S. faecium strains were confused with some of the, slowly reducing strains of S. faecalis. The reduction time of all known S. faecalis strains in ESD broth was less than 4 h. The optical density values, obtained from formazan extracts of cultures at 4 h, ranged approximately between 0.35 (strong TTC reducers, e.g., strain ATCC11700) and 0.12 (moderate Some of the colonies that developed on PSE failed to show the typical black halo of enterococci. The percentage of such atypical colonies varied from cheese to cheese; the lowest proportion that could be determined was 0% in the Kefalotyri sample and highest (51%) in the blue cheese sample. The recognition and enumeration of these atypical colonies on PSE, quite easy early in the incubation, became progressively difficult after 21 h as the black salts of hydrolyzed esculin surrounding typical colonies diffused through the agar. A meaningful comparison of colony sizes on all five selective media became possible after 48 h of incubation, when measurements could be made on KF, RSH, and SCR media. Table 2 indicates the average diameters of colonies that developed on each medium. Statistical analysis of the means (Student-Newman-Keuls test) disclosed that the colonies grown on KF, PSE, RSH, and SCR media did not differ in size significantly. However, the size differences between colonies grown on ESD and all other media were significant at the 95% level. Specificity of ESD for enterococci. A presumptive identification was carried out on 1,077 isolates. This total represented strains from all selective media and most of the cheese samples included ( Table 2). Thirteen strains (1.2%) showed a positive catalase test. Table 3 gives the distribution of strains that reacted positively in 4, 3, 2, 1, or none of the presumptive enterococcus tests. The results indicate that SCR, RSH, and ESD media selected the highest percentages of strains that reacted positively in all four presumptive tests (66, 60.1, and 58% respectively). In contrast, KF and PSE yielded low percentages (21.1 and 30.7, respectively). Substantially similar comparisions were made when the statistical evaluation was based on the results of only two of the four tests performed, i.e., reaction on BEM and tolerance to 6.5% NaCl; the combination of the 2 tests was found recently to be a reliable criterion for a presumptive identification of group D streptococci (6,8). A total of 936 other isolates from 18 cheese samples were obtained on ESD agar (Table 4). These strains were subjected to verification of identity. They were all gram-positive cocci and catalase negative; with some minor variations, they conformed to the Sherman's criteria. They could grow in the presence of bile salts and hydrolyzed esculin, and they demonstrated the group D antigen. Strains that reduced TTC on the ESD agar plates and showed TTC reduction in less than 4 h in ESD broth were confirmed as S. faecalis by the following pattern of biochemical characteristics: they could grow in the presence of 0.05% potassium tellurite; they could produce acid from sorbitol and mannitol, whereas they failed to produce acid from arabinose, melibiose, and raffinose; they grew well in folic acid assay medium without the addition of the cofactor; they utilized pyruvate, malate, and serine as energy sources; and they were unable to grow at 50 C. Strains that did not reduce TTC were confirmed at S. faecium by their intolerance to potassium tellurite, their production of acid from mannitol, arabinose and, with some exceptions, from melibiose. These strains required folic acid for growth. They could not utilize pyruvate, malate, and serine, and most of them were able to grow at 50 C. A few TTC-nonreducing strains showed these reactions but failed to ferment mannitol and arabinose. They were considered S. faecium var. durans (3). The enterococci of 8 of the 18 cheeses (Table 4) were also enumerated by using the membrane filter technique and ESD broth. The counts by this method were slightly higher than those obtained in the surface-inoculated ESD agar plates. This was. probably due to some cell loss that occurred during the spreading of inocula on the surface of the agar by means of glass rods. DISCUSSION S. faecalis shows strong TTC reduction, whereas S. faecium reduces TTC weakly (1). However, this presumptive differentiation can be equivocal due to variation in the intensity of TTC reduction among biotypes of S. faecalis and the occasional faint reactivity of S. faecium variants. Langston et al. (11), using Barnes' TG medium and method, were not able to distinguish atypical S. faecium strains, reducing TTC faintly, from S. faecalis. After 8 h of incubation the appearance of both types of cultures was similar. The same authors noticed that some differentiation could be made between the two biotypes of enterococci when observations of TTC reduction were made earlier than 8 h. In our study, the kinetics of reduction of ESD broth also provided a better criterion for differentiation than the mere ability to reduce TTC. The distinction between the two species (including atypical varieties of S. faecium) was attained in less than 4 h ( Table 1). Differentiation by ESD proved useful in the isolation and identification of enterococci from various cheeses (Tables 2 and 4). Of the five selective media used, ESD sup-ported the fastest rate of growth and the maximum size of colonies. Differential counts on this medium could be determined in 25 out of 26 cheeses analyzed as early as 17 h, whereas the relatively large size of the colonies made their enumeration and isolation convenient. Quantitatively, the selective counts on ESD at 48 h appeared roughly comparable to those on RSH and SCR, media well suited for isolating and enumerating enterococci in milk, cheese, and other dairy products (10,14,15). The previous observation that ESD allows good growth of a wide range of physiological types of enterococci (5) was coupled in this study with demonstration of a high degree of selectivity for this group of bacteria ( Table 3). Two of the selective media used, KF and PSE, showed too broad specificity, since they allowed the selection of large percentages of strains with a combination of characteristics atypical for enterococci. Both PSE and KF were found to lack in specificity towards enterococci previously (6,7). The present data indicate that the selectivity of KF and PSE is downgraded appreciably when enterococci are isolated from habitats such as cheese containing preponderant numbers of lactobacilli and related bacteria. In contrast, the suitability of ESD for such isola- a Symbols:+, More than 80% of colonies reduced TTC; -, more than 80% of colonies did not reduce TTC; ±, both reducing and nonreducing types were present in substantial numbers. tions was demonstrated in this study; the specificity of ESD for enterococci was shown to be equivalent to that of RSH and SCR media (Table 3). In comparison with other available media, the preparation of ESD agar presents the average laboratory with some inconvenience. Several component solutions are made up and sterilized separately. Their mixing requires aseptic conditions. The addition of hot agar solution and the lability of TTC demand attention and control of temperature. For adequate exclusion of cations, it is preferable to use deionized water and purified agar. Surface inoculation is more time consuming than preparation of pour plates. Most of these limitations, however, can be eliminated with the liquid version of the medium. Used in conjunction with the membrane filter technique, ESD broth presents the same convenience as other standard media utilized in this manner.

v3-fos

2020-12-10T09:04:22.640Z

1974-07-01T00:00:00.000Z

237230172

s2

Involvement of Vitamin B6 in the Dethiomethylation of Methionine by Rumen Microorganisms1 The dethiomethylation of methionine by a dialyzed extract obtained from the protozoa-rich fraction of rumen fluid is stimulated 2.5-fold by pyridoxal phosphate and strongly inhibited by deoxypyridoxine, a pyridoxal phosphate antagonist. These effects are not seen with undialyzed extracts or with whole rumen fluid. It is suggested that the anaerobic dethiomethylation of methionine by rumen microorganisms requires pyridoxal phosphate as a cofactor. It has been shown by Salsbury et al. (9, 10, 15) that methionine and several methionine analogues were extensively degraded by rumen microorganisms. Both S-methylcysteine and methionine were good substrates for dethiomethylation, with S-methylcysteine being the more reactive. In studies of dethiomethylation in this laboratory, several methionine analogues have been used to delineate a general substrate specificity for the dethiomethylation reaction. Results have indicated that substitution on the N, S, or C2 atoms of methionine decreases or prevents activity (10). plastic vials that contained 5 ml of scintillation cocktail (Phase combining system, Amersham/Searle). 7-ml vial was fitted with a plastic sleeve to permit counting in a liquid scintillation counter requiring 20-ml vials (Nuclear-Chicago, ISOCAP 300). Counts were taken at a discriminator setting of 4.5 to 150.0 keV (program 2B) for 2 min at 0.25 sigma error and repeated three times. Blanks were handled identically and subtracted from experi- mental values. Quench correction was accomplished by using an interal standard because of high chemical and color quench from rumen samples, protein, and strong NaOH solutions. The disintegrations per min- ute determined for the 5 volumes were averaged disintegrations per minute and the mean was multi- plied by 60 to determine the total disintegrations per minute in all the traps of each scrubber train. This gave a figure for total )nethanethiol production for each fermentation. Segal and Starkey (12) and Ruiz-Herrera and Starkey (8) have suggested that methionine is first deaminated and then dethiomethylated by aerobic bacteria. Bird (1) suggests that similar pathways may function in the rumen in spite of the anaerobic nature of the rumen environment. This assumption is not supported by the work of Salsbury and Merricks (10), who found that the methionine hydroxy analogue, oxidizable to the methionine keto analogue, was a poor substrate for dethiomethylation by rumen microorganisms, suggesting that in the rumen the oxidation of the amino group of methionine does not precede the dethiomethylation reaction. Walker and Nader (6,14) have reported that when [35S ]methionine was added to a rumen fluid fermentation, 51% of the 35S appeared in the H2S pool. However, no attempt was made to distinguish between H23"S and CH335SH. Ohigashi et al. (7) showed that an enzyme preparation isolated from Escherichia coli which was adapted to methionine degraded 'Delaware Agricultural Experiment Station Miscellaneous Paper 696, Contribution 13 of the Department of Animal Science and Agricultural Biochemistry, University of Delaware, Newark, Del. 19711. methionine to methanethiol and a-amino butyric acid. The E. coli enzyme preparation was stimulated both by anaerobic conditions and by pyridoxal plus adenosine 5'-triphosphate (the latter constituting pyridoxal phosphate). Pyridoxal phosphate is thought to be the cofactor most often involved in the metabolism of amino acids (2). Both homocysteine desulfhydrase and cystathionase employ pyridoxal phosphate as the enzyme cofactor in a gamma-elimination reaction (13). The bonds between pyridoxal phosphate and the apoenzyme, once formed, are difficult to cleave. Phosphorylase can be resolved into pyridoxal phosphate and protein by dialysis against a deforming buffer (imidazole) and a specific carbonyl reagent (cysteine) but not by dialysis against either of these reagents alone (2). This paper reports experiments done to determine whether pyridoxal phosphate is involved as a cofactor in the dethiomethylation of methionine by rumen microorganisms in vitro. A method is described for the preparation of an enzyme extract from a protozoa-rich fraction of rumen fluid that actively dethiomethylates methionine. MATERIALS AND METHODS Rumen fluid sample. Rumen fluid samples were taken from a mature, rumen-fistulated Guernsey cow at various stages of the lactation cycle. The cow wasfed silage, hay, and pasture ad libitum, plus a concentrate during lactation. Samples were taken just after feeding grain but before feeding silage, strained through cheesecloth, and maintained at 39 C. Mixed protozoal extract. Three liters of strained rumen fluid was allowed to stand at 39 C in a four-liter separatory funnel for 1 h, or until good separation of fractions occurred. When rumen fluid is Be AND METHIONINE DETHIOMETHYLATION allowed to stand under these conditions, it separates into three recognizable layers: a thick, green top layer containing fine plant material, bacteria, and trapped protozoa; a middle layer consisting of a turbid, straw-colored or greenish liquid containing bacteria and a very few of the small, active protozoa; and a thick, cream-colored bottom layer composed mainly of protozoa. The protozoa-rich fraction was withdrawn from the bottom of the funnel several times as the protozoa settled. Successive portions were collected in 200-ml screw-cap centrifuge tubes and refrigerated at 8 C until all collections were completed. All collections were pooled and then centrifuged at 1,000 x g for 10 min at 0 C, giving a semisolid pellet. The supernatant was discarded, and the pellet was suspended in a mineral buffer at pH 7.0 (11) and recentrifuged at 1,000 x g for 10 min at 0 C. The resuspension-recentrifugation procedure was repeated until a clear supernatant was obtained. The pellet was once again suspended in the mineral buffer and sonically treated for 4 min using a maximum setting (Bronson sonifier, LA 75). The sonified preparation was centrifuged at 10,000 x g for 60 min at 0 C, and the supernatant was designated as the mixed protozoal extract. Gas chromatographic system. A dual-column, hydrogen flame ionization gas chromatograph (F and M Scientific, model 810) was used with stainless-steel columns (0.25 inch by 12 feet; ca. 0.635 by 365.76 cm), packed with 10% silicone oil DC-200, on a 60to 80-mesh diatoport S support (Hewlett Packard, Avondale, Pa.). Hydrogen and air were maintained at 20 lb/in2 (20 x 703.1 kg/m I and 33 lb/in 2 (33 x 703.1 kg/mi, respectively. The carrier gas, helium, was maintained at 40 lb/in2 (33 x 703.1 kg/m ) with rotometers set at 2.0 for a flow rate of 47.2 ml/min. Both the injection port and the ionization detector were maintained at 300 + 10 C, and the column oven was kept at 60 + 2 C. All analyses were carried out isothermally. Portions (10 ml) of the mixed protozoal extract were incubated in 20-ml vials in the presence of 134 jimol of methionine. The vials were fitted with channel rubber stoppers to facilitate sampling by syringe and were maintained at 39 C in a water bath. Samples of the gas in the headspace above the mixed protozoal extract were obtained with a gas-tight syringe, and 250 Mliters was injected directly into the injection port. Headspace gas samples were taken periodically over a 4-h period, and results were quantitated on a disk chart integrator. Fermentation system, gas scrubber. Portions (30 ml) of the mixed protozoal extract were incubated in 60-ml serum vials in the presence of 1 ACi (17.86 nmol of [methyl-'4C]) of methionine obtained from Amersham/Searle, plus 4.08 gmol of pyridoxal phosphate or deoxypyridoxine, or with no vitamin B., analogue. The serum vials were fitted with channel rubber stoppers and two 17-gauge syringe needles. Tygon tubing was used to connect one needle to a nitrogen carrier gas source and the other needle to a gas scrubber train. The train consisted of three 10-ml portions of 1 N NaOH in test tubes (22 by 175 mm) connected in series by Tygon tubing. All treatments were in duplicate, and values reported are mean values. Incubation was for 4 h at 39 C. At the end of the incubation period, 10-ml portions of 6 N HCl were injected into the serum vials, the the mixtures were maintained at 39 C for another 15 min to permit the collection of residual methanethiol released by the acid. Liquid scintillation counting procedure. The three 10-ml portions of 1 N NaOH were pooled after each experiment, and five 500-Aliter volumes were placed in 7-ml plastic vials that contained 5 ml of scintillation cocktail (Phase combining system, Amersham/Searle). Each 7-ml vial was fitted with a plastic sleeve to permit counting in a liquid scintillation counter requiring 20-ml vials (Nuclear-Chicago, ISOCAP 300). Counts were taken at a discriminator setting of 4.5 to 150.0 keV (program 2B) for 2 min at 0.25 sigma error and repeated three times. Blanks were handled identically and subtracted from experimental values. Quench correction was accomplished by using an interal standard because of high chemical and color quench from rumen samples, protein, and strong NaOH solutions. The disintegrations per minute determined for the 5 volumes were averaged disintegrations per minute and the mean was multiplied by 60 to determine the total disintegrations per minute in all the traps of each scrubber train. This gave a figure for total ["C )nethanethiol production for each fermentation. RESULTS Evaluation of dethiomethylase activity. An experiment was carried out to determine the rate of methanethiol production by whole rumen fluid, the top layer from rumen fluid which had been incubated for about 1 h, and the bottom layer obtained under the same conditions (Fig. 1). When the amount of methanethiol which had accumulated after incubation for 160 min was taken to represent the relative activities of these three fractions, the bottom layer had an activity per unit volume (160 x 10 I integrator units) that was 10% of the corresponding activity of whole rumen fluid (1,600 x 103 integrator units). Because of differences in numbers of protozoa in the rumen fluid samples and in losses during preparation and storage of extracts, there were wide differences in activity between the individual mixed protozoal extracts used in this study. Consequently, comparisons have generally been limited to treatments within the same experiment. Nevertheless, to obtain an indication of the activity of the mixed protozoal extracts relative to whole rumen fluid, the results shown in Fig. 2 for a protozoal extract were compared to those shown in Fig. 1. Comparison of the very low activity of the washed-cell suspension with the much higher activity of the sonically treated extract suggests that the activity measured was Effect of vitamin B, on dethiomethylation by the mixed protozoal extract. Incubation of undialyzed mixed protozoal extract with methionine plus pyridoxal phosphate or methionine plus deoxypyridoxal did not yield good evidence for stimulation of the dethiomethylation reaction by pyridoxal phosphate or inhibition by deoxypyridoxine (Fig. 5). After dialysis of 80 ml of extract for 60 h against 5 liters of distilled water at 10 C, the activity of the extract was reduced, but stimulation by pyridoxal phosphate and inhibition by deoxypyridoxine could be demonstrated (Fig. 6). Effect of vitamin B, on dethiomethylation by whole rumen fluid. Previous experimentation (unpublished work from this laboratory) had shown that pyridoxal phosphate (Fig. 3) had little effect on dethiomethylation by rumen microorganisms in experiments with whole rumen fluid. To test the possibility that this lack of stimulation reflected a decreased uptake of the vitamin by rumen microorganisms when supplied as pyridoxal phosphate, an experiment was conducted with pyridoxal (Fig. 3) as a vitamin B, analogue addition to the reaction mixture. The experiment also included addition of the vitamin Be antagonist deoxypyridoxine (Fig. 3) as one of the treatments. It can be seen from Fig. 4 that neither deoxypyridoxine nor pyridoxal had any effect on the dethiomethylation reaction when whole rumen fluid was used. To obtain a more precise measure of the involvement of pyridoxal phosphate in dethiomethylation, [methyl-14C ]methionine was used as the substrate (1 uCi/30 ml of the mixed protozoal extract) and pyridoxal phosphate or deoxypyridoxine was added to the reaction mixture. In these experiments, measurements of [C~Inethanethiol production were used to determine enzyme activity. Table 1 shows the relative amounts of [140 Cmethanethiol produced by various 4-h experiments. A different mixed protozoal extract was used for each experiment, and each was dialyzed for 36 h against distilled water (80 ml of extract to 5 liters of water at 10 C) prior to incubation. The mixed protozoal extract used for experiment 1 was much more concentrated than those used for the other two experiments, and this is reflected in the values shown in the last column of Table 1. However, almost twice the amount of ['1C kmethanethiol was produced by the pyridoxal phosphate-treated preparation as by the untreated preparation in all three experiments. These findings give substantial support to the VOL. 28,1974 gas chromatographic data presented in Fig. 6, which suggest the involvement of pyridoxal phosphate in the dethiomethylation reaction. Further, the results obtained in experiment 3 (Table 1) indicate that deoxypyridoxine did indeed inhibit dethiomethylation relative to the control. DISCUSSION The crude enzyme preparation used in this study was prepared from the protozoa-rich fraction of rumen fluid and represented only a small part of the total methionine dethiomethylase activity of the whole rumen fluid. The decision to use this fraction was based on the assumption that the mixed protozoal extract would present a less complex array of demethiolases than would an extract prepared from whole rumen fluid. The very low dethiomethylase activity of the washed-cell suspension, when compared with the sonic extract prepared from it (Fig. 2) and with the fractions of rumen fluid (Fig. 1), suggests that this was indeed the case and that the activity was endogenous to the protozoa. It remains to be determined whether the activity lost during dialysis was similar to that studied or indicated the presence of different, more labile enzymes. Some of our more recent work (unpublished data) indicates that the dialyzed extract may contain more than one dethiomethylase. The absence of an appreciable treatment effect from the addition of pyridoxal phosphate or deoxypyridoxine when whole rumen fluid was used (Fig. 4) suggests that dethiomethylation by whole rumen fluid may be a different reaction than that of the mixed protozoal extract. However, the undialyzed mixed protozoal extract also failed to show inhibition by deoxypyridoxine or stimulation by pyridoxal phosphate (Fig. 5), and it was not until the preparation had been dialyzed that a treatment effect from added analogue could be demonstrated (Fig. 6). We interpret this to indicate that pyridoxal phosphate is tightly bound to the enzyme and cannot be displaced by deoxypyridoxine or added pyridoxal phosphate. Dialysis apparently releases some of the bound pyridoxal and permits the demonstration of the inhibitory or stimulatory effects of the analogues. It is possible that this interpretation, as well as the assumption that pyridoxal is not required for the bulk of methionine dethiomethylase activity shown by whole rumen fluid, could account for the lack of analogue effect with whole rumen fluid. The dethiomethylation reaction described in this report appears to resemble the one described by Ohigashi et al. (7), who suggested that a phosphorylated derivative of pyridoxal was required as a cofactor. Mitsuhashi (3), Mitsuhashi and Matsuo (4), Miwatani et al. (5), and Ohigashi et al. (7) reported that their enzyme preparations dethiomethylated methionine much more actively under anaerobic than under aerobic conditions. This may indicate that the mechanism(s) of dethiomethylation by these enzymes was such that they functioned more effectively at low oxygen tensions or that aerobic conditions favored destruction of the enzymes. Segal and Starkey (12) proposed a mechanism in which an initial oxidative deamination of methionine was followed by dethiomethylation of the a-keto--y-mercaptobutyric acid formed. This would exclude the possibility of involvement of pyridoxal phosphate as a cofactor in their system. Ruiz-Herrara and Starkey (8) have shown that a purified methionine dethiomethylase from Aspergillus utilized both methionine keto analogues and methionine hydroxy analogues as substrates. This would be expected if the dethiomethylation were preceded by deamination. We have found that methionine hydroxy analogues can be dethiomethylated by whole rumen fluid (9), although much less effectively than is methionine, but not by the mixed protozoal extract. The failure of the methionine hydroxy analogue to act as a substrate for the mixed protozoal extract is readily explained by the Bo AND METHIONINE DETHIOMETHYLATION requirement for the amino group in order to form a Schiff base with the pyridoxal moiety. The dethiomethylation of the analogue by whole rumen fluid may indicate that a portion of the dethiomethylase activity of the rumen fluid is attributable to an enzyme(s) not requiring pyridoxal as a cofactor, or it may reflect an initial amination of the methionine hydroxy analogue to form methionine, which then serves as a substrate for the pyridoxal-requiring dethiomethylase.

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2020-12-10T09:04:20.582Z

1974-12-01T00:00:00.000Z

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Agricultural Plants and Soil as a Reservoir for Pseudomonas aeruginosa Pseudomonas aeruginosa was detected in 24% of the soil samples but in only 0.13% of the vegetable samples from various agricultural areas of California. The distribution of pyocin types of soil and vegetable isolates was similar to that of clinical strains, and three of the soil isolates were resistant to carbenicillin. Pseudomonas aeruginosa multiplied in lettuce and bean under conditions of high temperature and high relative humidity (27 C and 80-95% relative humidity) but declined when the temperature and humidity were lowered (16 C, 55-75% relative humidity). The results suggest that soil is a reservior for P. aeruginosa and that the bacterium has the capacity to colonize plants during favorable conditions of temperature and moisture. Pseudomonas aeruginosa is currently considered one of the most frequent causative agents of hospital-associated infections and therefore has been the subject of many epidemiological investigations. Studies on the sources and vehicles by which the organism is transferred to patients have previously been limited to the immediate environment of patients inside the hospital. Such implicated sources include sinks, solutions and creams, hands of personnel, and inhalation and resuscitation equipment (4,5,9,12,13,16). Little attention was given to natural sources contributing to disease until Kominos et al. (10) found high counts of P. aeruginosa in raw vegetables in the hospital kitchen and suggested that these foods could act as primary vehicles for introducing the organism to patients. Their studies were motivated from the findings of Shooter et al. (15,16), who found that foods and salads in hospital kitchens contained Escherichia coli, P. aeruginosa, and Klebsiella species and showed that patients could acquire these bacteria in their intestinal flora. Ornamental potted plants (J. J. Cho, S. K. Green, M. N. Schroth, and S. D. Kominos, Phytopathology 63:1215, 1973 were also found to harbor P. aeruginosa, indicating that plants may serve as a natural reservoir in nature. Significant decrease of Pseudomonas infection in burn patients was noted by S. D. Kominos (unpublished data) following the elimination of fresh vegetables from the diet. The pyocin types of P. aeruginosa from vegetables were distributed similarly to those isolated from clinical specimens. The finding that consumption of vegetables in hospitals could be a means by which patients contract P. aeruginosa suggested a study to determine whether agricultural soils act as natural reservoirs of this bacterium. It also seemed important to investigate whether food plants are commonly colonized by P. aeruginosa from soil in the field, or if this might occur during harvest, or during a later processing stage, before reaching the consumer. This report described the occurrence of P. aeruginosa in agricultural soils and plants and the capacity of clinical isolates to survive in plants during various environmental conditions. MATERIALS AND METHODS Field isolations. Soil and vegetable material were sampled at five randomly selected sites from 58 different locations in various agricultural areas of central California during the summer of 1973. Soil samples from each field were collected to a depth of 30 cm, combined, and thoroughly mixed. Fifty grams of each soil sample was placed in 100 ml of sterile distilled water (pH 7.2) and shaken for 1 h at 300 rpm. A portion (0.5 ml) of each soil suspension was then added to 4.5 ml of broth consisting of acetamide and salts (17). Tenfold dilutions to 10-4 were made, and after 48 h of incubation at 42 C, 0.1 ml of each dilution was plated on King medium B (8) with 0.03% cetrimide added (KBC) (2), and the plates were incubated at 42 C. 987 The soils sampled in counties 1 to 5 were a sandy loam with pH values varying between 7.0 and 7.6. The soils sampled in county 6 were a loamy sand with a pH of 7.3. The irrigation water in counties 1, 2, 4 and 6 was from wells and in counties 3 and 5 from reservoirs. The water was delivered to the fields by pipes and was determined to be free of P. aeruginosa in all cases (30 samples) by the previously described isolation techniques. Fertilization of the fields was by inorganic fertilizers. Isolation from vegetable samples. In most instances, samples of plant material were taken from five different plants at five random sites per field so that a total of 25 plants was sampled from each field. The following parts were assayed: tomato, one friut and one leaf per plant; lettuce, five outer leaves and the heart per plant; celery, three stalks (two from outside, one from inside) and the heart per plant; cauliflower, two leaves and the head of one plant (three different plants were sampled at each random site); and spinach, one leaf per plant (10 different plants were sampled at each random site). The vegetable samples were sliced when necessary and placed individually into flasks containing phosphate-buffered (0.01 M) acetamide broth (pH 7.0). Isolation and identification. All cultures were incubated for 48 to 96 h at 42 C to allow growth of P. aeruginosa while inhibiting saprophytic bacteria (7). Cultures grown in acetamide broth and on KBC agar which fluoresced with an ultraviolet lamp were suspected to be P. aeruginosa. Representative yellow or blue-green fluorescent colonies from each sample were isolated on King medium B and confirmed as P. aeruginosa by the characterization scheme of Gilardi (6). Sensitivity of the isolation procedure. Samples of soil, a clay loam and a sandy loam with a pH of 7.0 and 7.2, respectively, were seeded with P. aeruginosa at a range of 1 to 10, 10 to 100, and 100 to 500 cells per 50 g of soil and processed as previously described. Lettuce, celery, and tomato fruits were similarly tested. Five replications were used per treatment. After 4 days of incubation, P. aeruginosa was recovered from all flasks seeded with P. aeruginosa at the 1 to 10 range. P. aeruginosa was not recovered from unseeded control flasks consisting of soil or vegetable material. Inoculation of plants. Lettuce and bean plants were inoculated with P. aeruginosa to determine whether or not the bacterium multiplied in plant tissues and to determine how long it persisted under different environmental conditions. A clinical strain of P. aeruginosa (PA 8) grown overnight on King B slants was used in all experiments. Leaves of lettuce plants (Lactuca sativa L., "Great Lake"), at the six-leaf stage, were infiltrated under vacuum with a cell suspension of 7 x 10' cells/ml of P. aeruginosa. This was done by placing the potted plants upside down in a vacuum chamber. The leaves were completely immersed in the inoculum, and the pots were held by a platform with aluminum foil placed across the soil surface to prevent soil loss. A vacuum of 710 mm of Hg was obtained, then released, at which time the leaves became thoroughly infiltrated with the inoculum, giving the leaves a watersoaked appearance. Control leaves were infiltrated with water. The plants were then grown in greenhouse chambers at 16 and 27 C, and at relative humidities fluctuating between 55 to 75% and 80 to 95%. The population of the bacterium was measured at 0, 1, 2, 3, 4, 6, 8, 10, 13, 16, 20, 25 and 30 days after inoculation by cutting one 16-mm disk from each of five infiltrated leaves per plant. The disks were rinsed three times in sterile distilled water to remove most of the bacteria adhering to the surface and then ground with a mortar and pestle in 5 ml of sterile distilled water. Three 0.1-ml portions from each dilution of a 10-fold dilution series were plated on KBC. Estimates of the bacterial populations were made after incubation at 42 C for 48 h. The above experiments were repeated five times, using three plants per treatment. Bean plants (Phaseolus vulgaris L., "Pinto") were infiltrated when the primary leaves were approximately 9 cm long. The plants were infiltrated with 7 x 10' cells of bacteria per ml and maintained under the same environmental conditions as the lettuce plants. Two 5-mm disks were cut from primary leaves of each plant and then processed as in the lettuce experiments. The experiments were repeated twice with three plants per treatment. To insure that any increase in bacterial population in the above experiment was a reflection of bacterial multiplication in the intercellular spaces of the plants and not on the plant surface, one disk from each of the previously described treatments was imprinted on KBC and removed. There was never more than five colony-forming units per disk except at the cut edges of the disks. As a further check to differentiate between multiplication of P. aeruginosa in the intercellular spaces and the leaf surface, the entire experiment with lettuce was conducted twice as previously described, with the exception that the leaf disks were immersed in 0.5% sodium hypochlorite for 1 min to surface sterilize them, then air-dried before plating on KBC. There was no apparent difference when comparing data from experiments where the disks were surface treated with sodium hypochlorite or washed with water. Pyocin typing. Strains of P. aeruginosa were differentiated by the method of Darrell and Wahba (3) as modified by Zabransky and Day (18). A pyocin producing isolate of P. aeruginosa was defined as a certain type by the inhibition produced against 11 indicator strains of P. aeruginosa. Sensitivity testing. Antimicrobial susceptibility to carbenicillin was determined by the disk diffusion method (1). RESULTS Pseudomonas aeruginosa was recovered from 24% (14 out of 58) of the soil samples (Table 1), and of these positive samples 71% (10 out of 14) were isolated from county 1. Soils in which tomatoes were grown yielded the highest frequency of isolation; P. aeruginosa was present in 45% (11 out of 24) of the tomato fields. Only 11% (1 out of 9) of the celery soils contained P. aeruginosa. The bacterium was not recovered from lettuce (0 out of 12) or cauliflower fields (0 out of 3). Of the remaining fields assayed, representing nine different crops in five counties, P. aeruginosa was only detected in one cotton and one corn field, both from county 6. Twenty-nine percent of the soils that were positive for P. aeruginosa contained more than 100 P. aeruginosa cells/g (dry weight) of soil (Table 2). Pseudomonas aeruginosa was recovered only twice from plant material collected from 43 fields ( Table 1). It was isolated from one tomato leaf from a field in which P. aeruginosa was detected in the soil and from one celery plant in a field where P. aeruginosa was not isolated from soil, even though a total of 10 random soil samples were taken, instead of the usual 5 (Table 1). Table 3 shows the pyocin types of P. aeruginosa isolated from soil and plants. Type D-2 and B-7 were the most frequently recovered, but nontypeable strains of P. aeruginosa were also commonly isolated from soil. Other types occasionally recovered were D-5, D-9, K, X-1 and X-10. The two types isolated from plants were B-7 from tomato and S from celery. Of these 34 isolates, 22 were sensitive to carbenicillin, 9 exhibited intermediate resistance, and 3 were resistant. Moderately high temperatures and humidities appeared to favor colonization and survival of P. aeruginosa within plant tissue (Fig.. 1). At 27 C and 80-95% relative humidity P. aeruginosa multiplied within the plant tissue and caused a rot of the leaves. The leaves began to yellow 2 to 3 days after inoculation, concomitant with bacterial multiplication. The bacterial population reached its highest level shortly before the leaves wilted (8 to 10 days). Waterinfiltrated plants maintained under the same conditions but not inoculated with the bacterium began to yellow approximately 13 days after treatment because of the unfavorable effect of high temperature and humidity on lettuce growth. When plants were maintained at high temperature and low humidity, the population of P. aeruginosa remained almost constant for about 15 days before slowly decreasing (Fig. 1). A slight yellowing of the infiltrated leaves was observed after 10 to 15 days. Control plants infiltrated with water showed no symptoms during the experiment. The population of P. aeruginosa decreased after infiltration to plants that were grown at 16 C, although the rate of decrease was considerably lower when plants were maintained at a high relative humidity (Fig. 1). At this low temperature, there was no evidence of injury to leaves infiltrated with bacteria or water when the plants were incubated at either high or low humidity. The survival and multiplication of P. aeruginosa within bean tissues (Fig. 2) under different environmental conditions were similar to that in infiltrated lettuce leaves. The leaves wilted after inoculation with bacteria when grown at high temperature and high humidity. Otherwise, there were no symptoms of injury. DISCUSSION Pseudomonas aeruginosa was recovered from 24% (14 out of 58) of the California soils tested, which suggests that it is fairly common soilborne bacterium in agricultural soils. That these soils are a natural habitat for the bacterium is supported by the facts that there was no history of organic fertilizers being used on the fields, no pasturing of animals, and irrigation water was free of the bacterium. Although various textbooks cite soil as a common source for the bacterium, we, as well as Ringen and Drake (14), could find little published data to support the statements. Furthermore, Ringen and Drake (15) found P. aeruginosa in only 3 of 100 soil samples and concluded that "its natural habitat, in part at least, is human feces and sewage." Our greater percentage of detection of P. aeruginosa from soil is presumably a reflection of improved isolation techniques. We have no explanation for the sporadic occurrence of P. aeruginosa in the different localities. It did not appear to be related to soil type, pH range, or fertilization practices. It was surprising that such a high percentage of soil was found to contain P. aeruginosa when considering the relatively few samplings per field. Many microorganisms are not evenly distributed throughout soil and occur in clumps or pockets of soil associated with a food base. This may be the case with P. aeruginosa, and thus its occurrence in soils could well be even more prevalent than indicated by our detection methods. Pyocin typing of P. aeruginosa showed that soil contained a variety of strains, many of which had the same pyocin production pattern as clinical strains. The most commonly isolated types of P. aeruginosa from clinical specimens, B-7 and D-2 (10), were also the types most frequently detected in soil. This suggests that soil constitutes a possible reservoir for strains of P. aeruginosa found in humans. The other types, D-5, D-9, K, X-1, X-10 and nontypables, have infrequently been recovered from clinical specimens (10). Of particular importance was the isolation of three carbenicillin-resistant strains of P. aeruginosa from soil. Carbenicillin-resistant strains have also been isolated from vegetables Growth of P. aeruginosa in leaves of pinto beans at various environmental conditions: 0, 16 C, 55-75% relative humidity; 0, 16 C, 85-95% relative humidity; 0, 27 C, 55-75% relative humidity; A, 27 C, 85-95% relative humidity. Each point represents six replications. served to patients (S. D. Kominos, unpublished data). Such resistant strains may be selected through antibiotic therapy and thus lead to further complications of the infectious process. Lowbury et al. (11) demonstrated the emergence of highly resistant strains of P. aeruginosa following carbenicillin treatment of hospitalized patients. Kominos et al. (10) showed that fresh vegetables commonly carried strains of P. aeruginosa and considered them to be a major vehicle for the dissemination of this bacterium. However, in this investigation, P. aeruginosa was only found twice in plants sampled in the fieldfrom a tomato leaf (type B-7) and from a celery plant (type S). It was surprising that the recovery of P. aeruginosa from the celery plant was in a field where it was not detected in the soil. Most likely, however, it was present in soil but was not detected. The rare finding of P. aeruginosa on vegetables in California, even though present in various soils, raises questions as to how the relatively high percentage of vegetables sampled by Kominos et al. (10) became colonized by P. aeruginosa. If plants are not widely colonized by P. aeruginosa in growing fields, as this study suggests, then contamination may occur during harvest, handling, processing, and transit. Vegetables may come in contact with soil, insects, humans, and other sources of P. aeruginosa, thereby acquiring the organism. Colonization would thus occur rapidly, if, during processing and transit, there were periods when temperature and moisture favored bacterial growth. This concept is strengthened by the findings of Kominos et al. (10), which showed that transported vegetables (especially tomatoes) contained large numbers of P. aeruginosa. The incidence of P. aeruginosa in plants and soils from California and other states should be studied for several years, however, before concluding that this organism is an infrequent colonizer of plant materials in farm lands. The environment differs substantially among years, and, as with common bacterial diseases of California plants, there are years when conditions do not favor bacterial infection. The California climate, in general, would not seem to be conducive for P. aeruginosa to colonize plants because of the semiarid conditions where most crops are grown. Climates where a high humidity is common, with frequent rainfall, would appear ideal for P. aeruginosa invasion of plants. Sprinkler irrigation would also appear to favor P. aeruginosa, since splashing water could carry the bacterium to above-ground plant parts where they could enter the plant through water-congested areas. All the fields tested in this study were furrow irrigated and situated in an area where the average summer rainfall was negligible.

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2020-12-10T09:04:20.753Z

1974-09-01T00:00:00.000Z

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Selecting Lysine-Excreting Mutants of Lactobacilli for Use in Food and Feed Enrichment Lysine analogues are used to select for lysine-excreting mutants of Lactobacillus plantarum. The use of lactobacilli that excrete lysine for the enrichment of foods and feedstuffs by fermentation is discussed. The increase in lysine content of soybean milk by a mutant of L. bulgaricus and in silage by L. plantarum is shown. Lysine analogues are used to select for lysine-excreting mutants of Lactobacillus plantarum. The use of lactobacilli that excrete lysine for the enrichment of foods and feedstuffs by fermentation is discussed. The increase in lysine content of soybean milk by a mutant of L. bulgaricus and in silage by L. plantarum is shown. Lysine, a limiting amino acid in both human and animal nutrition, is added to many foods and feedstuffs. If lysine could be incorporated into foods and feedstuffs in the course of natural fermentation by lactic acid bacteria, or yeasts (e.g., fermented dairy products, beer, or silage), considerable nutritional advantage would accrue. Bacteria regulate their amino acid biosynthesis so that they produce no higher concentrations of amino acids than they need for growth (3). However, mutants have been reported (3) which overproduce amino acids, generally because of defective repressor systems or inoperative feedback inhibition. We report a method to select and assay for spontaneous mutants that overproduce lysine. Lactobacillus plantarum, ATCC 8014, a common lactic acid producer, excreted less than 1 Mg of lysine per ml when grown in a lysine-free broth (lysine assay medium, Difco Laboratories, Detroit, Mich.). Cultures were incubated in shake culture for 24 h at 30 C, and cell number increased from 106 to 6 x 107. Growth was completely inhibited by the lysine analogue S-2-aminoethyl-L-cysteine (2; Sigma Chemical Co., St. Louis, Mo.) at concentrations as low as 5 Ag/ml. This inhibition was eliminated by L-lysine at about one-fifth the molar concentration of the analogue. To select. spontaneous lysine-excreting mutants, paper disks impregnated with 2 Mg of the analogue were placed on the surface of lysine assay medium solidified with 1.1% Ionagar (Colab Laboratories, Inc., Glenwood, Ill.) that had been seeded on the surface with L. plantarum. After 4 days of incubation at 30 C, spontaneous mutants of L. plantarum appeared within the zone of inhibition. The ability of these spontaneous mutants to excrete lysine was tested by streaking them on the surface of plates of solid lysine assay medium seeded with Leuconostoc mesenteroides ATCC 8042, a strain that requires lysine and is used in standard assays. We tested DL-a,Ediaminopimelic acid (Sigma Chemical Co., St. Louis, Mo.) of which the L form is the immediate precursor of lysine and found it would not support growth of L. mesenteroides. Colonies that provided maximal growth of the assay organism were isolated and subjected to mutation selection cycles with solid media. That is, each mutant was retested against a higher level (10 Ag/disk) of S-2-aminoethyl-L-cysteine, reisolated, and tested against a still higher level of the analogue. The cycle of testing and reisolation was repeated at least four times, until L. plantarum isolates resistant to a level of 5 mg of the analogue per disk were obtained. Lysine excretion of selected spontaneous mutants of L. plantarum and the increase with higher doses of S-2-aminoethyl-L-cysteine is shown in Table 1. The highest level of lysine excretion obtained was 72 l~g/ml, compared with less than 1 yg/ml for the wild type. Silage isolates of L. plantarum (obtained from M. K. Woolford, Grassland Research Institute, Hurley, Maidenhead, England) were grown on lysine assay medium and excreted less than 1 fig of lysine per ml, as was found with L. plantarum ATCC 8014. These silage isolates were also tested with S-2-aminoethyl-L-cysteine, as previously described, and lysineexcreting mutants were selected. Mutants were also tested against two other lysine analogues, L-lysine hydroxamate and fl-hydroxylysine, mixed DL and DL-allo (Sigma), and both were as effective as S-2-aminoethyl-L-cysteine. Mutants resistant to S-2-aminoethyl-L-cysteine were not necessarily resistant to other analogues, and vice versa. Inoculation of chopped fodder is successful in improving the final product (5). We inoculated lysine-excreting mutants of L. plantarum into small samples of freshly chopped maize plants. After 3 days of anaerobic incubation the samples of silage inoculated with wild-type L. plantarum contained 0.45% L-lysine (dry weight basis), whereas the mutant strain yielded 0.63% L-lysine. The method for selection of spontaneous lysine-excreting mutants might be used for lactic acid bacteria used in yoghurt, sour cream, buttermilk, fermented soybean milk, and sauerkraut, as well as silage. We selected lysine-excreting mutants of L. bulgaricus (wild type obtained from C. W. Hesseltine, Northern Regional Research Laboratory, Peoria, Ill.). The mutants and the wild type were inoculated into soybean milk (4). The mutants increased the lysine content of the fermented soybean milk from 4 to 14%. Mutant yeast might also be employed in brewing beer, and in fact a lysine-excreting yeast, grown on hydrocarbon, has been described (1). We thank Margaret Finkbeiner for technical assistance.

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2020-12-10T09:04:12.897Z

1974-05-01T00:00:00.000Z

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Campylobacter fetus Subspecies jejuni (Vibrio fetus) from Commercially Processed Poultry Three isolates of the human and animal pathogen Campylobacter fetus ss. jejuni (Vibrio fetus) were obtained from 165 poultry meat samples purchased from local retail stores. Campylobacter fetus (Vibrio fetus) is a causative agent of bovine and ovine abortion, avian hepatitis, scours in pigs and calves, as well as a cause of human infection. It has been isolated from the intestinal contents and organs of several meat animals, including cattle and sheep (1, 8), pigs (6), chickens, and turkeys (10). Dekeyser et al. (2) recovered the organism from mixed microflora (stools) in humans with diarrhea symptoms. Many investigators feel that the incidence of these infections in man is highly underrated. The mode of transmission of human C. fetus infection is obscure, particularly when the disease is found in individuals living in an urban environment without a history of animal contacts. Campylobacter fetus gastroenteritis in man has been traced to the milk supply (4), consumption of raw beef serum (3), and raw beef liver (11). The present study shows further evidence to indicate a possible food-borne epidemiology for human infection with C. fetus. Campylobacter fetus ss. jejuni was isolated from chicken meat obtained from retail stores (Table 1). This organism has not previously been reported from commercially processed chicken parts. The method of isolation and the selective media containing antibiotics were similar to those used by Shepler et al. (7) for the isolation of C. fetus from bull preputial fluid, and those used by Plastridge et al. (5) for the isolation from bull semen and Smibert (8,10) for the isolation from animal feces. The chicken parts were surface rinsed with nutrient broth (BBL, Cockeysville, Md.) by shaking in polyethylene bags. The broth was then filtered (0.65 ,um) and spread on the surface of a selective medium of Brucella agar (Pfizer, Inc., N.Y.) containing 2 units of bacitracin/ml, 2 gg of novobiocin/ml, and 1 unit of polymyxin/ml. These plates were incubated at 37 C for 3 to 5 days in a microaerophilic atmosphere (5% 0,, 'Present address: Route 1, Box 79, Pembroke, Va. 10% CO2, 85% N2). Suspect colonies were picked on the basis of being unpigmented and having a relatively small diameter (less than 2 mm). These were screened for nonfermentation of glucose, oxidase and catalase production, and morphological appearance under phase microscopy. Preparation of these media is described by Smibert (9). Further biochemical tests were employed to confirm the identity of these isolates (see Campylobacter in Bergey's Manual of Determinative Bacteriology, 8th ed., in press). Contaminants were a significant problem in the isolation and recovery of C. fetus from mixed poultry microflora. Alcaligenes faecalis was particularly annoying. This organism can be easily mistaken for C. fetus after the initial screening procedures since it has similar biochemical characteristics and morphological appearance. The difficulties encountered in the isolation of C. fetus and the selective method of recovery suggest that the incidence of this bacterium as found on retail poultry meat is a minimum value. The isolates were biochemically and morphologically indistinguishable from 12 other C. 995 fetus ss. jejuni strains previously isolated from human and avian disease. C. fetus organisms were shown to be capable of surviving on the surface of chicken meat at refrigeration (3 C) and freezing ( -23.5 C) temperatures for periods long enough to allow the meat to be marketed at the retail level (5 days at 3 C and 20 days at -23.5 C). Since the microorganism has been isolated from other meat animals (1, 6, 8), similar microbiological analyses of meat products such as pork, beef, lamb, or turkey may reveal C. fetus contamination. Appreciation is extended to J. Dekeyser, R. E. Weaver, and R. M. Smibert for supplying the C. fetus strains used in this study. This work was supported in part by funds from the Virginia Agricultufal Experiment Station.

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