Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).

Effect of Hydrogen Peroxide and Sodium Chloride on Enumeration of Thermally Stressed Cells of Staphylococcus aureus.

Evidence is presented suggesting that the decreased enumeration of heat-stressed Staphylococcus aureus cells on selective media is the result of accumulation of metabolic H2O2. It accumulates due to the decreased activity of catalase caused by the synergistic effects of heat and NaCl. Heated cells enumerated anaerobically on tryptic soy agar (TSA) containing 6.5% NaCl (TSAS 6.5) exhibited a 200-fold increase compared to cells enumerated aerobically on the same medium. The anaerobic counts on TSAS 6.5 were similar to the aerobic counts on TSA. Increases in both death and injury occurred when S. aureus was propagated in tryptic soy broth (TSB) plus 10% NaCl (TSBS) instead of TSB before thermal injury. Addition of catalase to TSA and TSA containing 7.5% NaCl (TSAS) increased the count to approximately the same levels on TSA and TSAS as that found following thermal injury after propagation in TSB. Catalase activity was 12-fold higher in stationary phase cells propagated in TSB than in TSBS. Indirect evidence indicates that toxic levels of H2O2 accumulated rapidly, causing one to two log decreases in enumeration after 30 to 60 min incubation on TSAS.

Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus.

The effects of the addition of catalase (EC 1.11.1.6) or pyruvate on the enumeration of Staphylococcus aureus in Trypticase soy broth with 10% NaCl were examined using a most-probable-number technique. Addition of catalase or pyruvate to the broth increased enumeration of all heat-stressed S. aureus strains tested. Increases were also observed with nonstressed cells. Catalase and pyruvate were similarly effective when added to Trypticase soy broth-10% NaCl in enumerating staphylococci naturally present in low-temperature-rendered ground-beef samples.

Catalase and enumeration of stressed Staphylococcus aureus cells.

The effects of catalase on the enumeration of stressed (heated, reduced water activity, or freeze-dried) Staphylococcus aureus cells on several selective media were examined. The addition of catalase greatly increased the enumeration of stressed cells. The beneficial effects of catalase were most pronounced on those media least efficient in enumeration of stressed staphylococci, showing increases in enumeration of up to 1,100-fold. The effects of catalase appear to be due to the reduced ability of stressed cells to repair and form colonies in the absence of an exogenous decomposer of H2O2. Thermally stressed cells were more sensitive to H2O2 than unstressed cells. During recovery, stressed cells overcame the requirement for catalase. These findings implicate H2O2 as a factor in the failure of certain selective media to adequately enumerate stressed cells and demonstrate that the addition of catalase to these media markedly increases their productivity.

Diluent sensitivity in thermally stressed cells of pseudomonas fluorescens.

Thermally injured cells of Pseudomonas fluorescens were unable to produce colonies on Trypticase soy agar (TSA) after dilution with 0.1% peptone. Nutritional exigency could not be used as the criterion for this injury, since varying the composition of the plating medium had little effect on the number of colonies that developed. The injured cells had no requirement for compounds known to leak out during the heat treatment in order to recover. The cells did not exhibit injury if dilution preceded heat treatment on the plating medium, demonstrating that the heat treatment sensitized the cells to the trauma of dilution. Substitution of 0.1% peptone with growth medium as the diluent largely offset the previously observed drop in TSA count. Little difference in survival was observed when monosodium glutamate or the balance of the defined medium was used as the diluent. The diluent effect was ionic rather than osmotic. The presence of cations was important in maintaining the integrity of the injured cell, and divalent cations enhanced this protective effect. The role of these cations at the level of the cell envelope is discussed.

Gentle lysis of Staphylococcus aureus at low temperature.

A low-temperature lysis procedure for Staphylococcus aureus is described. It is simple and requires no special equipment.

Catalase: its effect on microbial enumeration.

The addition of catalase to the surface of selective medium plates permitted increased enumeration of physically or chemically injured (stressed) microorganisms. Catalase acted by preventing the accumulation of hydrogen peroxide in, or around, injured cells. Heat-injured Staphylococcus aureus cells had decreased catalase activity, and heat-inactivated catalase had no effect on enumeration.

Cell-bound lipase and esterase of Brevibacterium linens.

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.

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.

Oxidative activation of Bacillus cereus spores.

A study was made of the activation of Bacillus cereus strain T spores by using the oxidizing agent sodium perborate. The degree of activation was measured with constant germination conditions by using L-alanine, inosine, adenosine, and L-alanine plus adenosine as germination stimulants. The germinal response following the various treatments was compared with the responses obtained with heat activation. It was concluded that the optimal time for activation with 30 mM sodium perborate at room temperature was about 4 hr. If the exposure time was greatly extended, the spores would germinate spontaneously. When the perborate treatment followed heat activation, the germinal response to L-alanine was stimulated, to inosine retarded and without apparent effect for adenosine or L-alanine plus adenosine. Results of experiments designed to demonstrate deactivation by slow oxidation showed that spores activated with sodium perborate were not deactivated by slow oxidation, whereas those activated by heat were. A deactivation study using mercaptoethanol as the deactivation agent showed that both methods of activation could be deactivated after a 24-hr exposure, but this deactivation was reversible by extending the exposure to mercaptoethanol. The results of heat-sensitivity studies revealed that about 70% of the sodium perborate-activated spores were heat sensitive after 60 min in a germination menstruum of L-alanine plus adenosine, whereas similarly treated heat-activated and nonactivated spores were about 99.99% heat sensitive, respectively.

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.

Interference-contrast and phase-contrast microscopy of sporulation in clostridium thermosaccharolyticum grown under strict anaerobiosis.

Cells of Clostridium thermosaccharolyticum grown under strict anaerobiosis (modified Hungate technique) were examined during growth and sporulation by employing Nomarski interference-contrast and Zernike phase-contrast optics to delineate the sequence of morphological changes leading to the formation of free, mature spores. A 0.5% l-arabinose, liquid, complex medium was used to obtain a yield of 30 to 40% free, refractile spores (ca. 10(8)/ml) by 48 hr of incubation. The mean doubling time for the glucose culture (vegetative cells) was found to be 80 min, and that for the l-arabinose culture (sporulating cells), 498 min. By 8 hr of incubation, beginning spore formation became evident in the arabinose culture by the development of a distinct arrowhead-shaped terminal swelling. By 32 hr of incubation or shortly thereafter, Nomarski optics showed the mature spore to be uniformly spherical, whereas the enlarged terminal swelling containing it was not. The use of phase-contrast and interference-contrast optics permitted the characterization of the distinctive morphological changes occurring during sporulation of C. thermosaccharolyticum.

Precursor ribosomal ribonucleic acid and ribosome accumulation in vivo during the recovery of Salmonella typhimurium from thermal injury.

When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA.

Biosynthesis during recovery of heat-injured Salmonella typhimurium.

Salmonella typhimurium 7136 incorporated label from glucose-U-(14)C into nucleic acids, lipids, and pool material during recovery from heat injury. There was very little incorporation of label into protein during recovery.

Requirements of Salmonella typhimurium for recovery from thermal injury.

The heating of Salmonella typhimurium 7136 at 48 C for 30 min produces a population of cells that are incompetent at division on Levine Eosin Methylene Blue Agar containing 2.0% NaCl (EMB-NaCl). When these injured cells were placed in fresh citrate salts medium they recovered, and regained their tolerance to the EMB-NaCl medium and grew out. The addition of the selective inhibitors rifamycin, 5-fluorouracil, 2,4-dinitrophenol, chlorotetracycline, chloramphenicol, and 5-methyl-tryptophan to the recovery medium showed that the recovery process was dependent on ribosomal ribonucleic acid (RNA) synthesis, adenosine triphosphate synthesis, and the synthesis of new protein. These results were substantiated by incorporation experiments, which demonstrated that during recovery no deoxyribonucleic acid synthesis, and hence no cell division, occurred. Ribosomal RNA was synthesized during recovery, but its synthesis was not the rate-limiting step. A small but significant amount of protein was also formed during the latter part of the recovery period.

Glycerol Ester Hydrolase Activity of Microbacterium thermosphactum.

Microbacterium thermosphactum possesses a significant glycerol ester hydrolase (lipase, EC 3.1.1.3) activity and a weak but definite carboxylic ester hydrolase (esterase, EC 3.1.1.1) activity. Harvested whole cell preparations contained 53 units of lipase activity with tripropionin as the substrate. This activity decreased with an increasing chain length of fatty acid in the triglyceride to 13 units with trilaurin as the substrate and no activity with tripalmitin. Maximum lipase activity was found at a temperature of 35 to 37 C and at a pH of 7.1 to 7.3. Lipase activity was associated with three different protein peaks when the protein of cell-free extract was fractionated by polyacrylamide gel electrophoresis.