APPLIED MICROBIOLOGY, Apr. 1975, p. 565-566
Vol. 29, No. 4 Printed in U.S.A.
Copyright 0 1975 American Society for Microbiology
Rhythmic Changes in Dry Heat Resistance of Bacillus subtilis Spores After Rapid Changes in pH ROBERT J. HECKLY* AND J. DIMATTEO Naval Biomedical Research Laboratory, School of Public Health, University of California, Berkeley, California 94720 Received for publication 21 October 1974
Heat resistance of freeze-dried Bacillus subtilis spores varied in a rhythmic function of time between acidification to about pH 1.5 and freezing. A comparable rapid shift to pH 11 produced little change in resistance to heat.
manner as a
It was previously shown (4) that survival of vegetative cells after lyophilization varied in a rhythmic manner if abrupt changes of their environment were imposed prior to freezing. The experiments reported in this note were designed to determine whether or not spores would exhibit a similar rhythmic response to a sudden change in environment. Because spores are considered by many to be nearly dormant, in that they react slowly to changes in environment (2), effects comparable to those observed with vegetative cells might not be obtained. Resistance of spores to dry heat was measured, rather than mere survival of dry organisms as was done previously with vegetative cells (4), because even the sensitive spores would probably have survived for months. Shifts in pH were selected because this is easily monitored, and it has been shown that acidification markedly affects heat resistance of spores (1, 2). The results of these experiments show that a rhythmic response can be induced in spores by at least one type of environmental change. The general procedure used was that previously described (1). The pH of a spore suspension was shifted by adding rapidly, with stirring, either 0.5 M HCl or 0.5 M NaOH to a cold, washed suspension of Bacillus subtilis var. niger spores in distilled water. This resulted in a pH change from 6.8 to 1.3 or from 6.8 to 11. At intervals, 0.5-ml samples were removed and frozen rapidly in bottles precooled in a dry ice bath. After drying overnight at pressures less than 0.02 torr, dry air (-70 C dew point) was admitted to the system, the bottles were stoppered in situ, and the stoppers were secured with a crimped aluminum cap. The bottles were then heated by complete immersion in vigorously boiling water for 30 min and cooled to room temperature. Viability of the spores was evaluated by the
usual procedures of making serial 10-fold dilutions immediately after reconstitution of the samples with 5 ml of distilled water and plating on Trypicase soy agar (BBL) using a drop method (5). Colonies were counted after 24 h of incubation at 31 C. The lower curve in Fig. 1 is typical of the results obtained after acidification to pH 1.3. In this experiment the spore suspension contained 5 x 10' colony-forming units (CFU)/ml before pH adjustment. Freezing and drying alone had a negligible effect on viability of the acidtreated spores, because lyophilized samples removed at 500 and 1,200 s after adding HCl contained 4.4 x 10" and 4.7 x 101 CFU/ml, respectively, when reconstituted without heating. Samples that were frozen 400 and 1,800 s after the pH shift were significantly more sensitive to heating than those frozen 900 and 1,650 s after the pH shift (Fig. 1). In no instance were comparable changes in heat sensitivity demonstrable after a shift to pH 11 (upper curve, Fig. 1). The small differences in the data points of this curve show that the precision of viability assays is good and that the variations shown after acidification cannot reasonably be ascribed to experimental errors. After reconstitution of acid-treated and lyophilized spores, they were uniformly sensitive to heat. Acidification of the spore suspension increased their sensitivity to dry heat, and one would expect that the trend established during the first minutes would have continued. However, it is clear that such is not the case. As with vegetative cells (4), the precise times of changes in resistance to heat was not predictable on a day-to-day basis. For example, in another experiment with the same batch of spores and in which every effort was made to have conditions identical with that represented in Fig. 1, the minima in resistance were at 250 and 1,750 s with high values from 565
566
NOTES
APPL. MICROBIOL.
academic interest in studies concerned with sterilization or decontamination procedures such as are proposed for use on space craft. It is also possible that heat resistance of spores used by the food-processing industry to evaluate the effectiveness of a sterilization procedure may be affected by environmental changes immediately prior to test. Anomalous data points, which have been interpreted to indicate recuperation, have been observed in culture preservation work (4). Since a variety of environmental changes stimulated rhythmic response in vegetative cells, it is possible that environmental shifts other than the drastic pH shifts used in these experiments could evoke a rhythmic response in spores. Therefore, it is important that any studies on surviving microorganisms, either vegetative or spores, include consideration of immediate history of the organism as well as the usual factors of cell age, nutrition, "protective" additive, pH, and moisture and oxygen content of the atmosphere.
vE r-
.E
-P
Time from pH shift to freezing
(Seconds\
FIG. 1. Effect of pH shifts on survival of B. subtilis var. niger. The pH was shifted abruptly at zero time to the indicated values, and samples were removed at intervals, frozen rapidly, and dried. After heating the dried cells for 30 min at 100 C, cells were reconstituted with distilled water.
750 to 1,500 s after the pH shift. Apparently this rhythmic response to a shift in environment is related to the dynamics of adaptability. A rhythmic response of Serratia marcescens to heating at 50 to 56 C in aqueous suspension was shown to be related to age or previous history of the cultures (3). The rhythmic response phenomenon is not trivial and may be of more than
This research was supported by the Office of Naval Research through a contract with the Regents of the University of California.
LITERATURE CITED 1. Alderton, G., and N. Snell. 1969. Chemical states of bacterial spores: dry-heat resistance. Appl. Microbiol. 17:745-749. 2. Alderton, G., P. A. Thompson, and N. Snell. 1964. Heat adaptation and ion exchange in Bacillus megaterium spores. Science 143:141-143. 3. Dimmick, R. 1965. Rhythmic response of Serratia marcescens to elevated temperature. J. Bacteriol. 89:791-798. 4. Heckly, R. J., R. L. Dimmick, and N. Guard. 1967. Studies on survival of bacteria: rhythmic response of microorganisms to freeze-drying additives. Appl. Microbiol. 15:1235-1239. 5. Miles, A. A., S. S. Misra, and J. 0. Irwin. 1938. The estimation of bactericidal power of the blood. J. Hyg. 38:732-749.
Vol. 29, No. 4 Printed in U.S.A.
Copyright 0 1975 American Society for Microbiology
Rhythmic Changes in Dry Heat Resistance of Bacillus subtilis Spores After Rapid Changes in pH ROBERT J. HECKLY* AND J. DIMATTEO Naval Biomedical Research Laboratory, School of Public Health, University of California, Berkeley, California 94720 Received for publication 21 October 1974
Heat resistance of freeze-dried Bacillus subtilis spores varied in a rhythmic function of time between acidification to about pH 1.5 and freezing. A comparable rapid shift to pH 11 produced little change in resistance to heat.
manner as a
It was previously shown (4) that survival of vegetative cells after lyophilization varied in a rhythmic manner if abrupt changes of their environment were imposed prior to freezing. The experiments reported in this note were designed to determine whether or not spores would exhibit a similar rhythmic response to a sudden change in environment. Because spores are considered by many to be nearly dormant, in that they react slowly to changes in environment (2), effects comparable to those observed with vegetative cells might not be obtained. Resistance of spores to dry heat was measured, rather than mere survival of dry organisms as was done previously with vegetative cells (4), because even the sensitive spores would probably have survived for months. Shifts in pH were selected because this is easily monitored, and it has been shown that acidification markedly affects heat resistance of spores (1, 2). The results of these experiments show that a rhythmic response can be induced in spores by at least one type of environmental change. The general procedure used was that previously described (1). The pH of a spore suspension was shifted by adding rapidly, with stirring, either 0.5 M HCl or 0.5 M NaOH to a cold, washed suspension of Bacillus subtilis var. niger spores in distilled water. This resulted in a pH change from 6.8 to 1.3 or from 6.8 to 11. At intervals, 0.5-ml samples were removed and frozen rapidly in bottles precooled in a dry ice bath. After drying overnight at pressures less than 0.02 torr, dry air (-70 C dew point) was admitted to the system, the bottles were stoppered in situ, and the stoppers were secured with a crimped aluminum cap. The bottles were then heated by complete immersion in vigorously boiling water for 30 min and cooled to room temperature. Viability of the spores was evaluated by the
usual procedures of making serial 10-fold dilutions immediately after reconstitution of the samples with 5 ml of distilled water and plating on Trypicase soy agar (BBL) using a drop method (5). Colonies were counted after 24 h of incubation at 31 C. The lower curve in Fig. 1 is typical of the results obtained after acidification to pH 1.3. In this experiment the spore suspension contained 5 x 10' colony-forming units (CFU)/ml before pH adjustment. Freezing and drying alone had a negligible effect on viability of the acidtreated spores, because lyophilized samples removed at 500 and 1,200 s after adding HCl contained 4.4 x 10" and 4.7 x 101 CFU/ml, respectively, when reconstituted without heating. Samples that were frozen 400 and 1,800 s after the pH shift were significantly more sensitive to heating than those frozen 900 and 1,650 s after the pH shift (Fig. 1). In no instance were comparable changes in heat sensitivity demonstrable after a shift to pH 11 (upper curve, Fig. 1). The small differences in the data points of this curve show that the precision of viability assays is good and that the variations shown after acidification cannot reasonably be ascribed to experimental errors. After reconstitution of acid-treated and lyophilized spores, they were uniformly sensitive to heat. Acidification of the spore suspension increased their sensitivity to dry heat, and one would expect that the trend established during the first minutes would have continued. However, it is clear that such is not the case. As with vegetative cells (4), the precise times of changes in resistance to heat was not predictable on a day-to-day basis. For example, in another experiment with the same batch of spores and in which every effort was made to have conditions identical with that represented in Fig. 1, the minima in resistance were at 250 and 1,750 s with high values from 565
566
NOTES
APPL. MICROBIOL.
academic interest in studies concerned with sterilization or decontamination procedures such as are proposed for use on space craft. It is also possible that heat resistance of spores used by the food-processing industry to evaluate the effectiveness of a sterilization procedure may be affected by environmental changes immediately prior to test. Anomalous data points, which have been interpreted to indicate recuperation, have been observed in culture preservation work (4). Since a variety of environmental changes stimulated rhythmic response in vegetative cells, it is possible that environmental shifts other than the drastic pH shifts used in these experiments could evoke a rhythmic response in spores. Therefore, it is important that any studies on surviving microorganisms, either vegetative or spores, include consideration of immediate history of the organism as well as the usual factors of cell age, nutrition, "protective" additive, pH, and moisture and oxygen content of the atmosphere.
vE r-
.E
-P
Time from pH shift to freezing
(Seconds\
FIG. 1. Effect of pH shifts on survival of B. subtilis var. niger. The pH was shifted abruptly at zero time to the indicated values, and samples were removed at intervals, frozen rapidly, and dried. After heating the dried cells for 30 min at 100 C, cells were reconstituted with distilled water.
750 to 1,500 s after the pH shift. Apparently this rhythmic response to a shift in environment is related to the dynamics of adaptability. A rhythmic response of Serratia marcescens to heating at 50 to 56 C in aqueous suspension was shown to be related to age or previous history of the cultures (3). The rhythmic response phenomenon is not trivial and may be of more than
This research was supported by the Office of Naval Research through a contract with the Regents of the University of California.
LITERATURE CITED 1. Alderton, G., and N. Snell. 1969. Chemical states of bacterial spores: dry-heat resistance. Appl. Microbiol. 17:745-749. 2. Alderton, G., P. A. Thompson, and N. Snell. 1964. Heat adaptation and ion exchange in Bacillus megaterium spores. Science 143:141-143. 3. Dimmick, R. 1965. Rhythmic response of Serratia marcescens to elevated temperature. J. Bacteriol. 89:791-798. 4. Heckly, R. J., R. L. Dimmick, and N. Guard. 1967. Studies on survival of bacteria: rhythmic response of microorganisms to freeze-drying additives. Appl. Microbiol. 15:1235-1239. 5. Miles, A. A., S. S. Misra, and J. 0. Irwin. 1938. The estimation of bactericidal power of the blood. J. Hyg. 38:732-749.
