Effect of Several Environmental Conditions on the "Thermal Death Rate" of Endospores of Aerobic, Thermophilic Bacteria

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1 APPLIED MICROBIOLOGY, Nov., 1965 Copyright 1965 American Society for Microbiology Vol. 13, No. 6 Printed in U.S.A. Effect of Several Environmental Conditions on the "Thermal Death Rate" of Endospores of Aerobic, Thermophilic Bacteria FUMIO YOKOYA AND GEORGE K. YORK Department of Food Science and Technology, University of California, Davis, California Received for publication 13 July 1965 ABSTRACT YOKOYA, FuMIo (University of California, Davis), AND GEORGE K. YORK. Effect of several environmental conditions on the "thermal death rate" of endospores of aerobic, thermophilic bacteria. Appl. Microbiol. 13: The composition of the recovery medium affected the apparent heat resistance of Bacillus stearothermophilus when the ph of the medium was 7.0 but not when the ph was 6.5. The rate of thermal death at 110 C was exponential. Deviations from exponential rates of thermal death during the initial phases of heating at 96 C were observed with endospores of B. coagulans under different conditions of sporulation. Additionally, the apparent heat resistance was influenced by the composition of the media used for sporulation and recovery and by the composition of the suspending menstruum. The presence of M sorbic in the suspending menstruum at ph 7.0 and the temperature of incubation of the cultures after heating did not affect the apparent heat resistance of B. coagulans. Several explanations are discussed for the observed deviations from exponential thermal death rates and the effect of the environment on the apparent heat resistance of B. coagulans. In studies of destruction of bacterial populations with heat, one criterion of death is the failure of the heated cells to reproduce when placed in a favorable environment for a reasonable time. A graph of the logarithms to the base 10 of the number of survivors of a bacterial population plotted against time of exposure to a constant lethal temperature has been variously termed "thermal death rate" curve (El Bisi and Ordal, 1956a; Lamanna and Mallette, 1959), "thermal destruction" curve (Lamanna and Mallette, 1959), and "survivor" curve (Ball and Olson, 1957; Schmidt, 1957). The chemical and physical conditions of the environment in which the heated cells are placed affects the apparent heat resistance of the cells as expressed by the thermal death rate (Murrel, Olson, and Scott, 1950; Schmidt, 1957). In addition to affecting the rate, changes in the environment can also alter the shape of the curve (Frank and Campbell, 1957; Humphrey and Nickerson, 1961; Rahn, 1943). The composition of the suspending menstruum in which the bacterial cells are heated also affects the apparent heat resistance of a population (Anderson, Esselen, and Fellows, 1949; Davis and Williams, 1948; Fabian and Graham, 1953; Frank, 1955; Shull and Ernst, 1962; Viljown, 1926). Further, the conditions of sporulation affect the apparent heat resistance of bacterial endospores and the shape of the "thermal death rate" curve (Amaha and Ordal, 1957; Curran, 1935; Esty and Williams, 1953; Sommer, 1930; Sugiyama, 1951; Williams, 1929). This paper is an attempt to relate the effects of several environmental conditions on the apparent heat resistance of endospores of Bacillus coagulans and B. stearothermophilus in hopes of gaining insight into the mechanisms of heat resistance of microorganisms. MATERIALS AND METHODS Microbes. B. coagulans strain 43P was originally isolated from spoiled, canned tomato juice. B. stearothermophilus strain 1518 was originally isolated from spoiled, canned peas. Media. Three media were used for obtaining spore crops of B. coagulans. Medium A: Proteose Peptone, 0.5%; yeast extract, 0.5%; glucose, 0.5%; K2HPO4, 0.4%; and agar, 1.5%. Medium B: Proteose Peptone, yeast extract, and glucose, each 0.5%; K2HPO4, 0.05%; manganese sulfate, 50 ppm; and agar, 2.0%. Medium C: unsalted tomato juice (6% total solids concentration), 10%; K2HPO4, 0.5%; and agar, 2%. The three media were adjusted with HCl or KOH after sterilization to give a final ph of 5.0. Four media were used to count the survivors of 993

2 994 YOKOYA AND YORK APPL. MICROBIOL. B. coagulans: Medium A, containing 1%O potato starch, and medium B and medium E, which contained tryptone, 0.5%; glucose, 0.5%, bromocresol purple, 0.004%; and agar, 1.5%. The media were adjusted to give final ph values of both 5.0 and 6.7. Sorbic was also added to counting media, in certain instances, to a final concentration of M (0.01%). Spore crops of B. stearothermophilus were obtained on nutrient agar containing M tris- (hydroxymethyl)aminomethane (Tris) buffer (medium F). This medium was adjusted to give a ph of 7.0 after sterilization. The survivors of B. stearothermophilus were enumerated on medium F and on Halvorson's counting medium D, which contained: peptone, 1%; yeast extract, 1%; NaCl, 0.5%; and agar, 2.0%. The media were adjusted to final ph values of 6.0, 6.5, and 7.0. Incubation. Both organisms were incubated aerobically for sporulation and enumeration of survivors. Spores of B. stearothermophilus were harvested after 10 days, and survivors were counted after 48 and 72 hr of incubation at 55 C. Spores of B. coagulans were harvested after 10 to 14 days of incubation at 55 C, depending upon the medium employed. Sporulation by B. coagulans was not observed on any of the media after 20 days of incubation at 37 C, although there was growth at this temperature. Survivors of B. coagulans were counted after 48 and 72 hr of incubation at 37, 45, and 55 C. Preparation of spore crops. After sporulation was observed by phase-contrast microscopy, the growth was washed from the agar surface with sterile deionized water and was shaken mechanically with glass beads (diameter, 2 mm). The suspension was then filtered through a layer of sterile cheesecloth and a thin layer of cotton. The cells passing through the filter were then washed twice with sterile deionized water and treated with lysozyme (El-Bisi et al., 1962). The suspension was then three times alternately washed and shaken mechanically with glass beads, after which no clumping and only a few vegetative cells were observed. Next, suspension was heated for 5 min at 80 C, centrifuged, and suspended in a small volume of sterile deionized water. When not used immediately, the suspension was stored at 0 C. Suspending menstrua for heating. The spore suspensions were heated in distilled deionized water, in M phosphate buffer (ph 7.0), in M phosphate buffer (ph 7.0), and in M phosphate buffer containing M sorbic (ph 7.0). Heating methods. Suspensions of endospores of B. coagulans were heated at 96 C and those of B. stearothermophilus, at 110 C. These temperatures were maintained by a thermostatically controlled oil bath, and fluctuations in temperature in the suspending menstruum, measured potentiometrically over a 3-hr period, ranged from 0.1 to 0.5 C. At 96 C, the suspensions were heated in a 500-ml, three-necked, Pyrex flask equipped with a stirrer and thermocouple. The flask, containing 150 ml of the suspending menstruum, was sterilized, cooled, and immersed in the oil bath until the temperature of the suspending menstruum was constant. A 1-ml amount of the spore suspension was added, which caused a brief (5 to 15 sec) decrease in the temperature of from 0.2 to 0.6 C. At intervals of 3 to 5 min, 1.0-ml samples were aseptically removed and transferred to 9.0 ml of sterile water previously cooled to 4 C in an ice bath. The suspensions were heated at 110 C in Pyrex tubes 3 inches (7.6 cm) long with an inside diameter of 0.8 mm and an outside diameter of 1.5 mm. The tubes were sealed at one end, sterilized, and -weighed before and after the introduction of approximately 0.03 ml of spore suspension. The tubes were then sealed, placed in a rack, and immersed in the oil bath. Approximately 60 sec elapsed before the temperature of the contents of the tubes became constant. Tubes were removed at 10- to 30-min intervals, immersed in ice water, wiped with soft tissue, placed in cold acetone to remove the oil, and washed in water. The tubes were then rinsed in sterile distilled water, placed in tubes containing 9.8 ml of sterile water, crushed with a sterile glass rod, and shaken. Calculations. The term chosen to express the "thermal death rate" or the apparent heat resistance was the "D value," which is defined as the time, in minutes, to reduce the population by 90% at a constant, lethal temperature. The D values were obtained from the slopes of the thermal death rate curves where D = 1/slope. Because the D value is based on the assumption of a constant and exponential rate of death, only the straightline portions of those curves showing deviation from linearity were used to determine the D values. The statistical analysis of variance was determined among the D values obtained with different environmental conditions (Snedecor, 1957). RESULTS AND DISCUSSION When endospores of B. stearothermophilus obtained by growth on medium F were heated at 110 C in neutral M phosphate buffer, and counted on media D and F, exponential rates of destruction were observed without deviation from linearity over a period of 180 min. The differences in the average D values obtained with the two counting media were significant at ph 7.0 but were not significant at ph 6.5 (Table 1). Growth did not occur when the heated spores were subcultured onto the three media at ph 6.0. This finding suggests that, although germination and outgrowth of heated spores of B. stearothermophilus are most sensitive to hydrogen ion, other chemical differences in the media influence the apparent heat resistance of this microorganism. Media D and F differ primarily in the concentration and source of growth factors (0.3% beef extract versus 1 % yeast extract) and in the

3 V'OL. 13, 1965 THERMAL DEATH RATE OF BACTERIAL ENDOSPORES 995 TABLE 1. Effect of composition of counting medium on the apparent heat resistance of endospores of Bacillus stearothermophilus 1518a ph of medium Counting medium Initial D Initial count/ml count/ml D min D 4.2 X X F 4.7 X X Variance, ** F NS Sporulated on medium F; heated at 96 C. NS, not significant; *, significant at greater than 1% level S li TIME, MIN FIG. 1. Effect of sporulating medium on shape of thermal survivor curve of endospores of Bacillus coagulans 43. Symbols: = medium A, ph 5.0; O = medium E, ph 5.0; = medium B, ph 5.0. Spores heated in phosphate buffer (ph 7.0) and survivors counted on medium B (ph 6.7). presence of a nonionic buffer. Medium D containls the higher concentrations of both peptone and source of growth factors, and its use as a counting medium results in lower D values. This result suggests that there are compounds present in these ingredients which are antagonistic to the germination and outgrowth of heated endospores, and the observation that there was no significant difference in the number of colonies arising on the two media when inoculated with unheated spores (Table 1) supports the idea of an antagonistic effect. Deviations from exyponential rates of thermal death during the initial phases of heating were observed with endospores ot B. coagulans under different conditions of sporulation (Fig. 1). Additionally, D values for B. coagularns were significantly influenced by the composition of sporulation and counting media (Table 2) and the suspending menstruum (Table 3). The interaction between the effects of the sporulating and counting media on the apparent heat resistance was statistically significant (Table 2), but the influence of the interaction of the suspending medium and the counting medium was not (Table 3). The shape of the curves obtained by plotting the logarithms of the survivors against time is a function of the composition of the medium on which the endospores were obtained (Fig. 1). The endospores from medium B, which contained less phosphate than medium A and to which manganese sulfate was added, showed a lag of approximately 2 hr during heating at 96 C, regardless of the medium used to count the survivors (Fig. 1 and 2). Conversely, the endospores from medium A exhibited initially higher rates of destruction at 96 C before the rate became constant (Fig. 1). These initially higher rates lasted from 15 to 25 min, depending upon the medium used for counting the survivors (Fig. 3). When tomato juice containing added phosphate (medium C) was used for sporulation, lags were also observed, but they were of shorter duration than those observed when medium B was used for sporulation (Fig. 1 and 4). Based on the assumption of an exponential rate of death, higher initial thermal death rates have been attributed to the presence of endospores of different innate resistances in a given population (Ball and Olson, 1957; El-Bisi and Ordal, 1956b). Frank and Campbell (1957) observed a curvi- TABLE 2. Effect of sporulation and counting medium composition on the apparent heat resistance of endospores of Bacillus coagulansa Counting medium D values (min) on sporulating medium Avg D for _counting A B, ph 5 E, media A B, ph B, ph E Avg D for sporulation media Variance, F ** ** a Suspending menstruum, M phosphate buffer (ph 7.0); heated at 96 C. Variance, F, for interaction of counting and sporulation media, ** (**, significant at greater than 1% level).

4 996 YOKOYA AND YORK APPL. MICROBIOL. TABLE 3. Effect of suspending menstrua and counting medium composition on the apparent heat resistance of endospores of Bacillus coagulans 43Pa Suspending menstruum (ph 7.0) ~~~~~~~~~~~~~~~~~med Counting medium type Counting ph Phosphate buffer Avg D for counting Distilled Water M X A b A A + starch A + starch B B E E Avg D for suspending menstruum Variance, F ** ** Variance, F, of interaction of suspending and counting media NS a Sporulated -on medium A; heated at 96 C. NS not significant; **, level. b D value (minutes). significant at greater than 1% Downloaded from TlblE,MIN FIG. 2. Effect of counting medium on survival of endospores of Bacillus coagulans 481? produced on medium B (ph 5.0). Counting media:o* = medium A, ph 5.0; 0 = medium E, ph 6.7; A = medium B, ph 5.0; and i\ = medium B, ph 6f.7. Heated at 96 C in X phosphate bu.ffer (ph 7.0). linear survival curve with B. coagulans 43P heated at C, but they were unable to obtain spores of different resis:tances by differential centrifugation, as has been done with other sporeformers (Yesair and Cameron, 1936). If this type of deviation from linearity is not caused by heterogenous populations, then the rate of death is not constant and D values based on the assumption of an exponential rate are not valid. A change from initially higher rates to initially lower rates of thermal dlestruction resulted from a -J (I) 0 z o l TIME,MIN FIG. 3. Effect of counting medium on survival of endospores of Bacillus coagulans 48P produced on medium A (ph 5.0). Counting media: * = medium A, ph 5.0; 0 = medium E, ph 6.7; A = medium B, ph 5.0; and A = medium B, ph 6.7. Heated at 96 C in X phosphate buffer (ph 7.0). change in the composition of the media on which endospores were obtained. This suggests a shift from a heterogenous to a homogenous popula- on September 13, 2018 by guest

5 VOL. 13, 1965 THERMAL DEATH RATE OF BACTERIAL ENDOSPORES 997 -j 0 LA- z 2 50 on oo,5 0~~~~~~~~~~~~ FIG.04.- eo onigmeimo uvvlo L0 080 Ig0 TI ME, MIN FIG. 4. Effect of counting medium on survival of endospores of Bacillus coagulans 43P produced on medium C (ph 5.0). Counting media:@ = medium A, ph 5.0; 0 = medium E, ph 6.7; * = medium B, ph 5.0; A = medium B, ph 6.7. Heated at 96 C in M phosphate buffer (ph 7.0). tion of endospores with respect to heat resistance caused by changes in the physiology or biochemistry, or both, of sporulation. If, for example, manganese is required for the formation of "normally heat resistant" endospores of B. coagulans, a medium without added manganese would be depleted of this element during sporulation and the spores produced in its absence would not have the same resistance to heat. An initial lag or lower initial rates of thermal destruction can most often be explained by experimental errors, for example, lags in heat penetration, clumping of cells, protection of spores by vegetative cells, and contamination by spores of a different bacterial type. Several explanations have been proposed for initially slower rates when observed experimental errors have been eliminated. Initial lag times in thermal death have been attributed to such causes as the activation of germination of endospores by heat (Curran and Evans, 1944, 1945; Finley and Fields, 1962; Morrison and Rettger, 1930), the heat inactivation of reproduction initiators (El-Bisi and Ordal, 1956b; Humphrey and Nickerson, 1961), the inactivation of germination inhibitors, and the inactivation of inhibitors of reproduction initiators (El-Bisi and Ordal, 1956a). These explanations assume an accumulated effect of heat, with a constant rate of death occurring only after the inhibitors or initiators present in the spores have been subjected to or have absorbed a given amount of heat. If the thermal stability of such intrinsic mechanisms can be 'altered by the conditions under which sporulation occurs, this could explain the differences observed in the duration of the lag times with different sporulating media (Fig. 1). Alteration of the composition of the counting media, however, should not affect the heat stability of such intrinsic mechanisms and, thus, should not change the duration of the lag time. Although there was some alteration of the lag time with different counting media it was much less than the change observed with sporulating media (Fig. 1, 2, and 4). The effect of the counting medium was most pronounced on the number of endospores of B. coagulans germinating and then growing after various times of heating, which is reflected in the D values obtained from the straight-line portions of the survivor curves (Table 2). The effect of each counting medium on the apparent heat resistance of B. coagulans was equivalent, regardless of the medium used for sporulation; that is, medium E at ph 6.7 resulted in higher D values than medium B at 6.7, and medium A at ph 5.0 TABLE 4. Effect of incubation temperature on the apparent heat resistance of endospores of Bacillus coagulans heeated at 96 Ca Counting medium Incubation temp Type ph 37 C 45 C 55 C A b A B B E E Avg D for counting medium Avg D for incubation temp ** Variance, F NS a Sporulated on medium A; heated at 96 C. NS, not significant; **, significant at greater than 1% level. b D value (minutes).

6 998 YOKOYA AND YORK APPL. MICROBIOL. TABLE 5. Effect of sorbic (0.001 M) on the apparent heat resistance of endospores of Bacillus coagulans 43P* Counting medium Suspending (ph 7) media Sporulation medium Phosphate Sorbic Type ph buffer,, x x B B t 47.2 B B + sorbic B + sorbic B B + sorbic B B + sorbic B + sorbic * Incubated at 45 C; heated at 96 C. t D values (minutes). gave higher D values than medium B at ph 5.0 (Fig. 2 and 3). Higher D values were observed with B. coagulans when 1% starch was added to counting medium A at both ph 6.7 and 5.0 (Table 2), but the shape of the survivor curve was not affected (Fig. 4). The temperature at which the heated endospore populations were incubated did not cause large variations in the apparent heat resistance of B. coagulans (Table 4). The presence of M (0.011 %) sorbic in the suspending menstrua at ph 7.0 did not affect the apparent heat resistance of B. coagulans (Table 5). Spores obtained on medium B with and without M sorbic (ph 5.0) had similar heat resistances, and the presence of M sorbic in medium B used for counting did not cause a significant decrease in the D values (Table 5). Sporulation of B. coagulans was inhibited, however, in the presence of M sorbic in medium B (ph 5.0). LITERATURE CITED AMAHA, M., AND Z. J. ORDAL Effect of divalent cations in the sporulation medium on the thermal death rate of Bacillus coagulans var. thermourans. J. Bacteriol. 74: ANDERSON, E. E., W. B. ESSELEN, AND C. R. FELLOWS Effect of solids, salts, sugars and other food ingredients on thermal resistance of Bacillus thermourans. Food Res. 14: BALL, C. O., AND F. C. W. OLSON Steriization in food technology, 1st ed. McGraw-Hill Book Co., Inc., New York. CURRAN, H. R The influence of some environmental factors upon the thermal resistance of bacterial spores. J. Infect. Diseases 66: CURRAN, H. R., AND F. R. EVANS Heat activation inducing germination in the spores of thermophilic aerobic bacteria. J. Bacteriol. 47:437. CURRAN, H. R., AND F. R. EVANS Heat activation inducing germination in the spores of thermotolerant and thermophilic aerobic bacteria. J. Bacteriol. 49: DAVIS, F. L., JR., AND 0. B. WILLIAMS Studies on heat resistance. I. Increasing resistance to heat of bacterial spores by selection. J. Bacteriol. 66: EL-BISI, H. M., AND Z. J. ORDAL. 1956a. The effect of sporulation temperature on the thermal resistance of Bacillus coagulans var. thermourans. J. Bacteriol. 71: EL-BIsi, H. M., AND J. Z. ORDAL. 1956b. The effect of certain sporulating conditions on the thermal death rate of Bacillus coagulans var. thermourans. J. Bacteriol. 71:1-9. EL-BISI, H. M., R. M. LECHoWIcH, J. AMAHA, AND Z. J. ORDAL Chemical events during death of bacterial endospores by moist heat. J. Food Sci. 27: EsTy, J. R., AND C. C. WILLIAMS Heat resistance studies. I. A new method for the determination of heat resistance of bacterial spores. J. Infect. Diseases 34: FABIAN, F. W., AND H. T. GRAHAM Viability of thermophilic bacteria in the presence of varying concentrations of s, sodium chloride and sugars. Food Technol. 7: FINLEY, N., AND M. L. FIELDS Heat activation and heat-induced dornancy of Bacillus stearothermophilus spores. Appl. Microbiol. 10: FRANK, H. A The influence of cationic environments on the thermal resistance of Bacillus coagulans. Food Res. 20: FRANK, H. A., AND L. L. CAMPBELL, JR The nonlogarithmic rate of thermal destruction of spores of Bacillus coagulans. Appl. Microbiol. 5: HUMPHREY, A. E., AND J. T. R. NICKERSON Testing thermal death data for significant nonlogarithmic behavior. Appl. Microbiol. 9: LAMANNA, E., AND M. F. MALLETTE Basic bacteriology, 2nd ed. The Williams and Wilkins Co., Baltimore. MORRISON, E. W., AND L. F. RETTGER Bacterial spores. I. A study in heat resistance and dormancy. J. Bacteriol. 20: MURREL, W. G., A. W. OLSEN, AND W. J. ScoTr The enumeration of heated bacterial spores. II. Experiments with Bacillus species. Australian J. Sci. Res. 3: RAHN, The problem of the logarithmic order of death in bacteria. Biodynamica 4:

7 VOL. 13, 1965 THERMAL DEATH RATE OF BACTERIAL ENDOSPORES 999 SCHMIDT, C. F Thermal resistance of microorganisms. In G. F. Reddish [ed.], Antiseptics, disinfectants, fungicides, and sterilization, 2nd ed. Lea and Febiger, Philadelphia. SHULL, J. J., AND R. R. ERNST Graphical procedure for comparing thermal death of Bacillus stearothermophilus spores in saturated and superheated steam. Appl. Microbiol. 10: SNEDECOR, G. W Statistical methods applied to experiments in agriculture and biology, 5th ed. The Iowa State College Press, Ames, Iowa. SOMMER, E. W Heat resistance of spores of Clostridium botulinum. J. Infect. Diseases 46: SUGIYAMA, H Studies on factors affecting the heat resistance of spores of Clostridium botulinum. J. Bacteriol. 62: VILJOWN, J. A Heat resistance studies. II. The protective affect of sodium chloride on bacterial spores heated in pea liquor. J. Infect. Diseases 39: WILLIAMS, 0. B The heat resistance of bacterial spores. J. Infect. Diseases 44: YESAIR, J., AND E. J. CAMERON Centrifugal fractionation of heat resistance in a spore crop. J. Bacteriol. 31:2-3. Downloaded from on September 13, 2018 by guest