Radiation resistance and injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25 degrees C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and -30 degrees C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at -20 degrees C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at -20 degrees C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at -20 degrees C, nor did storage at -20 degrees C alter the cell's resistance to irradiation at 25 degrees C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36 degrees C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36 degrees C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5 degrees C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36 degrees C for 1 day than at 5 degrees C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Factors Affecting Comparative Resistance of Naturally Occurring and Subcultured Pseudomonas aeruginosa to Disinfectants

A strain of Pseudomonas aeruginosa was isolated in pure culture from the reservoir of a hospital mist therapy unit by an extinction-dilution technique; its natural distilled water environment was used as a growth and maintenance medium. After a single subculture on Trypticase soy agar, the strain showed a marked decrease in resistance to inactivation by acetic acid, glutaraldehyde, chlorine dioxide, and a quaternary ammonium compound when compared with naturally occurring cells grown in mist therapy unit water. The following factors were observed to affect the relative resistances of naturally occurring and subcultured cells of the P. aeruginosa strain: (i) temperature at which the cultures were incubated prior to exposure to disinfectants, (ii) growth phase of the cultures at the time of exposure to disinfectants, (iii) nature of the suspending menstruum for disinfectants, and (iv) exposure to fluorescent light during incubation of inocula prior to testing. The applied significance of these findings may alter the present concepts of disinfectant testing as well as routine control procedures in the hospital environment.     

Effect of reducing agents on oxidation-reduction potential and the outgrowth of Clostridium botulinum type E spores.

Oxidation-reduction potential (Eh) levels were measured and standardized to pH (Eh7) for Trypticase soy broth containing various concentrations of reducing agents. Prereduced Trypticase soy broth with no added reducing agents exhibited a potential of -141 mV. Ascorbic acid at 0.2 to 0.005% and sodium thioglycolate at concentrations below 0.05% produced an Eh7 higher than the prereduced Trypticase soy broth containing no added reducing agents. The addition of cysteine hydrochloride,2-mercaptoethanol, and sodium formaldehyde sulfoxylate to prereduced Trypticase soy broth resulted in a reduction of Eh7 compared to the system without added reducing agents. The order of relative reducing intensity (from highest to lowest) for the reducing agents when comparing molar concentration was: sodium formaldehyde sulfoxylate,2-mercaptoethanol, cysteine hydrochloride, sodium thioglycolate, and ascorbic acid. Optimal growth of the test organism occurred at low Eh7 and low concentration of the reducing agents. A direct correlation existed between growth of the test organism and -Eh7 x -log concentration of the reducing agent.     

Effect of reducing agents on oxidation-reduction potential and the outgrowth of Clostridium botulinum type E spores.

Oxidation-reduction potential (Eh) levels were measured and standardized to pH (Eh7) for Trypticase soy broth containing various concentrations of reducing agents. Prereduced Trypticase soy broth with no added reducing agents exhibited a potential of -141 mV. Ascorbic acid at 0.2 to 0.005% and sodium thioglycolate at concentrations below 0.05% produced an Eh7 higher than the prereduced Trypticase soy broth containing no added reducing agents. The addition of cysteine hydrochloride,2-mercaptoethanol, and sodium formaldehyde sulfoxylate to prereduced Trypticase soy broth resulted in a reduction of Eh7 compared to the system without added reducing agents. The order of relative reducing intensity (from highest to lowest) for the reducing agents when comparing molar concentration was: sodium formaldehyde sulfoxylate,2-mercaptoethanol, cysteine hydrochloride, sodium thioglycolate, and ascorbic acid. Optimal growth of the test organism occurred at low Eh7 and low concentration of the reducing agents. A direct correlation existed between growth of the test organism and -Eh7 x -log concentration of the reducing agent.     

Evaluation of Two Commercially Available Media for Detection of Bacteremia

Analysis of the results of 13,162 blood cultures during a 9-month interval has shown that Pseudomonas aeruginosa statistically was recovered more frequently from Trypticase soy broth (TSB) than from Thioglycollate-135C and that contaminants, including Staphylococcus epidermidis and aerobic and anaerobic Corynebacterium species, were isolated with statistically greater frequency from Thioglycollate-135C than from TSB. No other statistically significant differences were found.     

Pyrolysis gas-liquid chromatography of the genus Bacillus: effect of growth media on pyrochromatogram reproducibility.

Pyrolysis gas-liquid chromatography was performed on dried Bacillus microorganisms to evaluate the effects of growth media. Six cultures of Bacillus and six lot numbers of Trypticase soy agar (BBL) were used to test the hypothesis that a microorganism grown on various lot numbers of the same chromatogram. Also tested was the effect of three different media on chromatogram reproduction using the same six cultures. Results show little or no differences observed between the chromatograms of the individual Bacillus spp. grown on the six lot numbers of Trypticase soy agar. When chromatograms of the three different media were compared, several differences were observed, particularly in the areas most characteristic of individual species. Pryolysis gas-liquid chromatography can be a useful tool for the characterization or identification of the genus Bacillus if the chromatographic and cultural conditions are maintained.     

Agarose soy casein digest medium for replacement of blood agar for potency determinations of live Pasteurella vaccines.

Blood agar, prepared with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) soy agar and 5% defibrinated bovine blood, is used for testing the potency of live Pasteurella multocida and Pasteurella haemolytica vaccines, but its potential for variation makes it undesirable to use in a standard assay method. Tests done with RPMI 1640 and Trypticase soy medium indicated that the benefits obtained by adding defibrinated blood to the Trypticase soy agar medium were more likely due to neutralization of toxic components than to the presence of transferrin or iron as growth factors. Reduction of toxic components in the Trypticase soy agar medium was accomplished by replacing agar with agarose and by autoclaving glucose as a separate solution to produce the replacement medium. The replacement medium was prepared by autoclaving three separate solutions--Trypticase soy broth without glucose; glucose; and agarose--cooling to 55 degrees C, and mixing and then pouring the mixtures into petri dishes. The growth obtained with this medium as judged by determination of the number of CFU and the colony sizes of P. multocida or P. haemolytica was equal to or better than those obtained with blood agar.     

Agarose soy casein digest medium for replacement of blood agar for potency determinations of live Pasteurella vaccines

Blood agar, prepared with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) soy agar and 5% defibrinated bovine blood, is used for testing the potency of live Pasteurella multocida and Pasteurella haemolytica vaccines, but its potential for variation makes it undesirable to use in a standard assay method. Tests done with RPMI 1640 and Trypticase soy medium indicated that the benefits obtained by adding defibrinated blood to the Trypticase soy agar medium were more likely due to neutralization of toxic components than to the presence of transferrin or iron as growth factors. Reduction of toxic components in the Trypticase soy agar medium was accomplished by replacing agar with agarose and by autoclaving glucose as a separate solution to produce the replacement medium. The replacement medium was prepared by autoclaving three separate solutions--Trypticase soy broth without glucose; glucose; and agarose--cooling to 55 degrees C, and mixing and then pouring the mixtures into petri dishes. The growth obtained with this medium as judged by determination of the number of CFU and the colony sizes of P. multocida or P. haemolytica was equal to or better than those obtained with blood agar.     

Growth of Desulfovibrio on the Surface of Agar Media

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Induction and resuscitation of viable but nonculturable Salmonella enterica serovar typhimurium DT104

Salmonella enterica serovar Typhimurium DT104 11601 was tested for its ability to maintain viability in minimal, chemically defined solutions. Periodic monitoring of growth and survival in microcosms of different ion concentrations, maintained at various temperatures, showed a gradual decline in culturable organisms ( approximately 235 days) at 5 degrees C. Organisms maintained at a higher temperature (21 degrees C) showed continuous, equivalent CFU per milliliter ( approximately 10(6)) up to 400 days after inoculation. Fluorescence microscopy with Baclight revealed that nonculturable cells were actually viable, while observations with scanning electron microscopy showed that the cells had retained their structural integrity. Temperature upshift (56 degrees C +/- 0.5, 15 s) of the nonculturable organisms (5 degrees C) in Trypticase soy broth followed by immediate inoculation onto Trypticase soy agar (TSA) gave evidence of resuscitation. Interestingly, S. enterica serovar Typhimurium DT104 from the microcosms at either 5 degrees C (1 to 200 days) or 21 degrees C (1 to 250 days) did not show enhanced growth after intermittent inoculation onto catalase-supplemented TSA. Furthermore, cells from 21 degrees C microcosms exposed to oxidative and osmotic stress showed greater resistance to stresses over increasing times of exposure than did recently grown cells. It is possible that the exceptional survivability and resilience of this particular strain may in part reflect the growing importance of this multidrug-resistant organism, in general, as a cause of intestinal disease in humans. The fact that S. enterica serovar Typhimurium DT104 11601 is capable of modifying its physiological characteristics, including entry into and recovery from the viable but nonculturable state, suggests the overall possibility that S. enterica serovar Typhimurium DT104 may be able to respond uniquely to various adverse environmental conditions.     

Effect of Soy Proteins on the Growth of Clostridium perfringens

Proteins that are used to fabricate imitation foods such as synthetic meats were evaluated for stimulative or inhibitory effects on the growth of Clostridium perfringens. Growth rate and extent were measured in thioglycolate medium without dextrose. This liquid medium contains Trypticase (BBL) which served as the protein control. For comparison, various soy proteins, synthetic meats, beef, turkey, sodium caseinate, and combinations of each were substituted for Trypticase. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an E(h) of below -300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N(2)-10% CO(2)) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease.     

Fermentation of Peptides by Bacteroides ruminicola B(1)4

The maximum growth rate of Bacteroides ruminicola B(1)4 was significantly improved when either Trypticase or acetate and C(4)-C(5) fatty acids were added to defined medium containing macrominerals, microminerals, vitamins, hemin, cysteine hydrochloride, and glucose. The organism was unable to grow with peptides as the sole energy source, but growth yields from glucose were significantly improved when Trypticase was added to batch cultures containing basal medium, acetate, and C(4)-C(5) volatile fatty acids. During periods of rapid growth, very little peptide was deaminated to ammonia, but after growth ceased there was a linear increase in ammonia. Fifteen grams of Trypticase per liter resulted in maximum ammonia production. In glucose-limited chemostats, ammonia production from peptides was inversely proportional to the dilution rate, and 87% of the variation in ammonia production could be explained by retention time in the culture vessel. Chemostats receiving Trypticase had higher theoretical maximum growth yields and lower maintenance energy expenditures than similar cultures not receiving peptide. Cells from the Trypticase cultures contained more carbohydrate, and this difference was most evident at rapid dilution rates. When corrections were made for cell composition and the amount of peptides that were fermented, it appeared that peptide carbon skeletons could be used for maintenance energy. B. ruminicola B(1)4 was unable to grow on peptides alone because it was unable to utilize peptides at a fast enough rate to meet its maintenance requirement.     

Biochemical Alternations in Bacillus megaterium as Produced by Aflatoxin B1

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 μg of aflatoxin B₁ per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 μg of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.     

Morphological Alterations in Bacillus megaterium as Produced by Aflatoxin B1

Morphologically abnormal cells were produced by Bacillus megaterium NRRL B-1368 in response to aflatoxin B(1). Filamentous forms were characterized by early granulation and unusually large and numerous deposits of poly-beta-hydroxybutyric acid within the cells. Pantoyl lactone was without effect as a reversing agent for the observed inhibition of cell septum formation. B. megaterium cells and spores produced on toxic (3.8 mug of aflatoxin B(1) per ml) and nontoxic Trypticase Soy Broth and Trypticase Soy Agar (TSA) were observed by using phase contrast and electron microscopy. Transfer of aberrant forms to nontoxic TSA yielded macrocolonies with daughter cells morphologically indistinguishable from untreated cells. Agar slide cultures of filamentous cells transferred to nontoxic TSA indicated that normal cells were formed. Electron photomicrographs showed a decreased number of mesosomes in filamentous cells as compared to control cells. There were no observable morphological differences in spores formed on toxic or nontoxic TSA.     

Comparison of Media for Detection of Fungi on Spacecraft

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay.