Revival and Subsequent Isolation of Heat-Injured Bacteria by a Membrane Filter Technique

Cultures containing mixed flora from raw milk were heated at 62.8 C for 15, 20, 25, and 30 min. Dilutions were filtered through membrane filters, and the filters were incubated on Trypticase soy broth (TSB) and on TSB plus NaCl (TSBS). The TSB count indicated the total population which survived heating and included injured and uninjured cells. The colonies on TSBS indicated the uninjured cells and were marked by perforating the membrane near the colony. This membrane was then transferred to fresh TSB and incubated further. The injured organisms recovered and formed colonies which could be distinguished from previous colonies of uninjured organisms. Transfer counts on TSB were not substantially different from the initial TSB counts at 15, 20, 25, and 30 min of heating.     

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.     

Effect of environmental conditions during heating on commercial spore strip performance.

Commercial biological indicator spore strips in glassine envelopes, produced by three manufacturers, were evaluated by fraction-negative procedures after being heated at 121.0 +/- 0.05 degrees C. Only one type of spore strip met the manufacturer's specifications. The strips of one manufacturer were further evaluated by fraction-negative and survivor curve-plate count procedures after being heated under several conditions (enclosed in glassine envelopes, in trypticase soy broth plus 0.0015% bromocresol purple, in Trypticase soy broth alone in Water for Injection, directly); Trypticase soy broth plus bromocresol purple and tryptic soy agar, respectively, were used as recovery media. The heating condition affected the D-value of the spore strip. Recovery procedures also had an effect; in all cases, the D-values obtained from the survivor curve tests were larger than those obtained from fraction-negative tests carried out under the same conditions. To determine if the differences in D-values between the two evaluation procedures were caused by the recovery media, we evaluated, by both methods, one type of spore strip heated directly and in glassine envelopes, using tryptic soy agar plus bromocresol purple and Trypticase soy broth plus 1.5% agar, respectively, as the recovery media. The survivor curve results showed that for both enclosed and unenclosed spore strips, there was a marked difference between the two recovery media; however, there was no difference when fraction-negative tests were used.     

Characterization of Mild Thermal Stress in Pseudomonas fluorescens and Its Repair

The exposure of exponentially growing Pseudomonas fluorescens P7 cells to heating at 36 C for 2 h in a defined medium, followed by cooling to 25 C and further incubation at this, the optimal growth temperature, resulted in the apparent death of approximately 99% of the cells, as determined by their inability to form colonies on Trypticase soy agar. Continued incubation at 25 C resulted in an extremely rapid increase in the Trypticase soy agar count, demonstrating that the phenomenon observed was not death but rather injury. Presumptive evidence of heat-stimulated ribonucleic acid (RNA) degradation and membrane damage was provided by the observed loss of 260-nm absorbing materials. Confirmation of RNA degradation was obtained by colorimetric analysis. Ribosomal RNA from normal and injured cells, which was electrophoretically separated on polyacrylamide gels, revealed that the 23S and 16S species were only partially destroyed. Inhibitor studies demonstrated, however, that RNA synthesis was necessary for recovery. The unusual accumulation of 17S RNA during recovery pointed to the presence of a heat-induced lesion in the RNA maturation process. A thermally induced membrane lesion is also discussed.     

Effect of aeration on minimal medium recovery of heated Salmonella typhimurium.

The effect of presence or absence of air on minimal medium recovery of heated Salmonella typhimurium was investigated. It was determined that the expression of minimal medium recovery is not only dependent on heat and a nutritionally complex medium but also on air. Unlike in the presence of air, in the presence of nitrogen, cells were able to recover their ability to grow on Trypticase soy agar enriched with 0.5% yeast extract (TSY) when incubated in TSY broth. It was established that in the presence of nitrogen the number of heat-TSY- induced, single-straneded breaks in deoxyribonucleic acid (DNA) were less than in the presence of air. Furthermore, the DNA breaks in nitrogen were repaired, whereas DNA breaks in air were not. The ability of cells to grow on TSY agar corresponded well with their ability to repair damage to DNA.     

The vitamin requirements of Staphylococcus cohnii

In heat-resistance studies with spores of Clostridium sporogenes BC-2, an improved recovery medium was needed for severely heat-damaged spores as the used previously--Wynne medium in Miller-Prickett tubes--did not allow accurate counts of spores because of gas formation and disruption of agar. Initial test with pour plates of Viande-Leyure medium containing egg-yolk gave much increased counts for spores previously heated for 50 min at 112 degrees C; this increase was attributed to the presence of egg-yolk. Addition of egg-yolk to Reinforced Clostridial Agar, All-Culture Medium and Trypticase Soy Agar showed that Trypticase Soy Agar with egg-yolk was the best recovery medium. For the final formulation, the value of supplementation with cysteine-HCl and methylene blue was also shown. The resultant Egg-yolk Trypticase Soy Agar is conveniently prepared from BBL Trypticase Soy Agar (40 g) with the addition of 0.4 g/l cysteine-HCl, 4 mg/l of methylene blue and 2% Oxoid egg-yolk emulsion aseptically to the melted basal medium. For optimal spore counts, pour plates are incubated anaerobically for 5--7 d at 30 degrees C.     

Destruction of Staphylococcus aureus during frankfurter processing.

We studied the thermal resistance of Staphylococcus aureus during frankfurter processing in respect to whether staphylococci are killed by the heating step of the process and whether heat injury interferes with the quantitative estimation of the survivors. With S. aureus 198E, heat injury could be demonstrated only when large numbers of cells (10(8)/g) were present and at a product temperature of 140 degrees F (60 degrees C). On tryptic soy agar and tryptic soy agar plus 7% NaCl media, at temperatures less than 140 degrees F, the counts were virtually identical; above 140 degrees F, the counts converged, with the organisms dying so rapidly that heat injury was not demonstrable. Heat injury was thus judged not to interfere with the quantitative estimation of staphylococci surviving the normal commercial heating given frankfurters. By using a combination of direct plating on tryptic soy agar and a most-probable-number technique, we detected no viable cells (less than 0.3/g) of several strains of S. aureus in frankfurters heated to 160 degrees F (71.1 degrees C). This temperature is compatible with the normal final temperature to which federally inspected processors heat their frankfurters and with the temperature needed to destroy salmonellae.     

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.     

Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

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.     

Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

Thermal Injury and Recovery of Bacillus subtilis

Exposure of Bacillus subtilis NCTC 8236 to sublethal temperatures produced a change in the sensitivity of the organism to salt and polymyxin. After 30 min at 47 C, 90% of the population was unable to grow on a modified sulfite polymyxin sulfadiazine agar containing an added 1% NaCl, 1% glucose, and 1% asparagine. The data presented demonstrate that thermal injury results in degradation of both 16S and 23S ribonucleic acid (RNA) and in damage to the cell membrane, suggested by leakage into the heating mestruum of material absorbing at 260 nm. When the cells were placed in a recovery medium (Trypticase soy broth), complete recovery, indicated by a returned tolerance to salt and polymyxin, occurred within 2 hr. The presence of a protein inhibitor (chloramphenicol) and cell wall inhibitors (vancomycin and penicillin) during recovery had no effect, whereas the presence of an RNA inhibitor (actinomycin D) effectively inhibited recovery. Further data demonstrated that the injured cells were able to resynthesize both species of ribosomal RNA during recovery by using the fragments which resulted from the injury process. Also, precursor 16S and precursor 23S particles accumulated during recovery. The maturation of the precursor particles during recovery was not affected by the presence of chloramphenicol in the recovery medium.     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization.

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

Thermal injury of Yersinia enterocolitica

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Thermal injury of Yersinia enterocolitica.

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

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.     

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.