Heat-Induced Temperature Sensitivity of Outgrowing Bacillus cereus Sporest

APPLED AND ENVRONMENTAL MCROBOLOGY, Apr. 1984, p. 768-774 99-224/84/4768-7$2./ Copyright 1984, American Society for Microbiology Vol. 47, No. 4 Heat-nduced Temperature Sensitivity of Outgrowing Bacillus cereus Sporest KATHERNE M. JOHNSONt AND F. F. BUSTA* Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 5518 Received 22 August 1983/Accepted 18 January 1984 nactivation of Bacillus cereus spores during cooling (1 C/h) from 9 C occurred in two phases. One phase occurred during cooling from 9 to 8 C; the second occurred during cooling from 46 to 38 C. n contrast, no inactivation occurred when spores were cooled from a maximum temperature of 8 C. nactivation of spores at a constant temperature of 45 C was induced by initial heat treatments from 8 to 9 C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 C inactivation to occur; however, faster germiniation was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 3 or 35 C. Microscopic evaluation of spores outgrowing at 45 C revealed that when inactivation occurred, outgrowth halted at the swelling stage. nhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 C inactivation mechanism. Examination of Bacillus cereus spore survival during heating and cooling demonstrated that spore response could not always be predicted with data generated at constant temperatures (14). After rapid heating (ca. 9 C/h) to 9 C, spores were inactivated in two distinct temperature ranges during cooling at rates of 5 or 1 C/h. Thermal inactivation occurred during cooling from 9 to 8'C; the population remained stable during cooling fromn 8 to 5 C; and a second period of inactivation occurred during cooling from 5 to 35 C. nactivation that occurred at the lower temperatures was not observed when spores were heated slowly (2 or 4 C/h) to 8 C before cooling (16a). This phenomenon was observed with three of four B. cereus foodborne illnessrelated strains studied. The apparent low temperature inactivation of spores observed during cooling from 9 C must be viewed with more than academic curiosity. f the inactivation represents injury rather than death of spores, potentially viable spores may remain undetected with standard microbiological analysis. Repair of injured spores in food and subsequent growth may lead to potentially hazardous situations (3). Temperature sensitivity of B. cereus spores at 45 to 47 C has been reported (1, 5, 6). Strains with temperature sensitivity are generally isolated by mutagenic techniques such as treatment with nitrosoguanidine (1). Mutant spores with defective germination systems, RNA synthesis, protein synthesis, DNA synthesis (5), and cell development and division systems (6) during outgrowth have been observed. These strains exhibited normal vegetative growth at permissive (3 to 35C) and nonpermissive (45 to 47 C) outgrowth temperatures. Heating is known to induce a number of changes in spores through the process of activation (2). Faster germination rates, less exacting germination requirements, increased * Corresponding author. t Paper no. 13,574 of the Scientific Journal Series of the Agricultural Experiment Station, University of Minnesota, St. Paul. t Present address: Department of Food Science and Nutrition, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456. 768 metabolic activity (17), and changes in spore proteins and enzymes (29) have been reported. Activation has also been shown to cause apparent increases in populations of some spore suspensions (12). Germination is a degradative process that has been extensively reviewed (11). During germination, spores lose heat resistance and refractility. Proteins (24) and RNA are degraded to provide components for subsequent synthesis occurring during outgrowth (26). Outgrowth of spores is a highly ordered process (26, 3). Synthesis of RNA commences, followed shortly thereafter by protein synthesis and finally by DNA synthesis. Enzymes are synthesized in a specific order and only for limited periods of time (34). The appearance of germinating and outgrowing spores under phase-contrast microscopy also follows an ordered progression. Spores turn from phase bright to dark during germination; then swell, elongate, and divide during outgrowth (3). Due to the systematic patterns in spore outgrowth, morphological observations may provide some insight into metabolic functions involved in inhibition. n this paper, we intend to show that initial heat treatments can induce subsequent temperature sensitivity in B. cereus spores during outgrowth. This sensitivity is expressed both during cooling and at a constant temperature of 45 C. Attempts to repair damage and speculation on the metabolic function involved in heat-induced temperature sensitivity will be made. (This paper was presented at the 43rd Annual Meeting of the nstitute of Food Technologists, New Orleans, La., 19 through 22 June 1983.) MATERALS AND METHODS Spore preparation. B. cereus F481/72 spores were prepared on fortified nutrient agar and stored in distilled water at 4 + 2 C as previously described (15). Enumeration procedure. Samples were diluted in.1% peptone water and plated on plate count agar (Difco Laboratories) by the pour plate technique (8). Plates were incubated at 3 C for 24 + 4 h before enumeration of colonies. nitial trials indicated that 24-h plate count agar counts of stressed spores were equal to those on mannitol-egg yolk-polymyxin

VOL. 47, 1984 HEAT-NDUCED OUTGROWTH SENSTVTY N B. CEREUS 769 agar (19) and greater than those on Trypticase soy agar (BBL Microbiology Systems) at 24 or 48 h (data not shown). Germination and outgrowth measurements. Germination was evaluated by one of two procedures. Loss of heat resistance was employed to monitor germination rates. Samples (1 to 2 ml) were transferred to 13- by 1-mm tubes and heated at 7 C for 15 min before plating. Microscopic examination was used in studies on germination and outgrowth. A drop of spore suspension was dried on a clean glass slide at ca. 65 C. Dried samples were subsequently rehydrated and examined at 1,x magnification with a phase-contrast microscope (Nikon). One hundred spores were scored as phase bright, phase dark, swollen, elongated, dividing (3), or ghost. A ghost had the appearance of a hollow shell. Judgment was required to distinguish one stage from another; therefore, intermediate stages (e.g., between swollen and elongated stages) were occasionally enumerated. Generally, the order in which sample slides were examined was randomized to remove bias, and each slide was examined twice. Heat activation and inoculation procedures. Unless otherwise indicated, spores were heat activated at 7 C for 15 min in distilled water. Test media were held at <3C during inoculation for <5 min to prevent germination. Germination and survival during cooling. Heat-activated spores (16/ml) in Trypticase soy broth (TSB; BBL) were heated rapidly to 9 C and cooled to 1 C at a rate of 1 C/h as previously described (16a). Viability and germination (loss of heat resistance) were monitored periodically during cooling. The same procedure was used for samples heated rapidly to 8 C before cooling. Treatment of spores at constant temperatures. Heat-activated spores in TSB were heated at 9 C for 2 min and then transferred to water baths at 35, 45, or 55 C. Surface plating on mannitol-egg yolk-polymyxin was used for enumeration for samples immediately after heating and after 1 and 2 h of incubation. Two tubes for each treatment were evaluated at each sampling time. Germination and survival in L-alanine and phosphate buffer at 45 C. Sodium phosphate buffer (36 mm, ph 7.) with and without 1 mm L-alanine (L-Ala and buffer, respectively) were sterilized by autoclaving (121 C for 15 min). Tubes of buffer and L-Ala (.9 ml) at 45 C were inoculated with heatactivated spores (.1 ml) to achieve ca. 17 spores per ml. Germination (loss of heat resistance) and survival were monitored periodically. Variation of initial heat treatment in TSB. Heat-activated spores in TSB (ca. 16 spores per ml) were heated at 7, 8, 82.5, 85, 87.5, or 9 C for 15 min. Heating at 85 to 9 C caused inactivation of a portion of the spore population. After all heat treatments (i.e., 7 to 9 C), tubes were held at 7 C for an additional 15 min. This incubation period stopped inactivation that occurred at 85 to 9 C and reduced the potential of sampling error caused by temperature/ volume ratios. Tubes were then transferred to a 45 C water bath to initiate low temperature inactivation. Germination (loss of heat resistance) and viability were measured periodically. Each tube of TSB was used for one sample only. This entire segment of work was duplicated. nhibition of outgrowth by inhibitors of macromolecular synthesis. Minimum inhibitory concentrations of rifampin, chloramphenicol, and nalidixic acid for B. cereus were determined previously to be.5, 5, and 1,ug/ml (data not shown). nhibitor solutions were filter sterilized, prepared weekly, and stored at 4 + 2C, with the exception of nalidixic acid, which was prepared daily. ndividual tubes of TSB (1 ml) containing one of each of these inhibitors were tempered at 3 or 45 C before inoculation with heat-activated spores (ca. 17/ml). Samples were periodically removed from each tube; the population was enumerated or germination and outgrowth were evaluated microscopically (or both). Before inoculation, spore suspensions contained >95% refractile spores. Outgrowth of spores in TSB at 45 C after differing heat activation treatments. The effects of three different heat activation treatments on survival and simultaneous outgrowth were evaluated. The heat activation treatments evaluated were intended to elicit differing survival patterns at 45 C. n the first, spores were heat activated at 75 C for 15 min before inoculation of TSB (1 ml, ca. 17 spores per ml) at 45 C. n the second, TSB (1 ml) at.3c was inoculated (ca. 17 spores per ml) and heated at 8 C for 15 min before transfer to 45 C. n the third, TSB (1 ml) at <3C was inoculated with heat-activated spores (ca. 16/ml), heated at 87.5 C for 15 min, held at 7 C for 15 min, and finally incubated at 45 C. Survival and outgrowth were evaluated periodically by enumeration and microscopic procedures, respectively. Evaluation of potential injury. Heat-activated spores in L- Ala (ca. 17 spores per ml) were incubated for 2 h at 45 C to induce damage. The suspension was then vortexed, and.1 ml was delivered into 9.9 ml of potential repair medium previously tempered at 3 or 35 C. The dilution factor was sufficient to stop L-Ala germination after transfer (data not shown). Components initially screened as potential repair agents at 35 C included.1% peptone,.1% beef extract,.1% soluble starch, 2% sucrose,.25% glucose,.25% dipotassium phosphate,.1% calcium lactate,.5% sodium pyruvate, and.2% magnesium sulfate; 1 mm L-Ala was used as a control. Further studies were conducted at 3 C in duplicate with peptone, beef extract, starch (single trial), L- Ala, and TSB with or without inhibitors of macromolecular synthesis to insure that observed increases in numbers were not the result of multiplication of cells. Penicillin G (5 U/ml; Pfiezer) was used in addition to the inhibitors mentioned above. Statistical analysis. The linear portions of 45 C survivor curves in TSB after various initial heat treatments were analyzed by least-squares regression analysis. Data from 15 to 26 min were included in regression analysis. Log transformations of the percentage of survivors were used to obtain the best estimates and 95% confidence intervals of slopes (inactivation rate constants) and intercepts. These kinetic constants were subjected to analysis of variance by using VAN (33) and regression analysis of slope (logl scale) or intercept versus temperature by using MULTREG (32). Differences among the means of factors which had significant F-tests were tested by using the method of least significant differences (28). RESULTS AND DSCUSSON Germination and survival of spores during cooling. Spores heated rapidly to 9 C and cooled at a rate of 1 C/h exhibited two phases of inactivation as described previously (16a). No germination was apparent until the second phase of inactivation commenced at 44 to 46 C (Fig. 1). Germination was more rapid and more extensive than inactivation during cooling. Spores heated only to 8 C, however, maintained a constant population throughout the cooling process (Fig. 2). These data suggest that the lack of low-temperature inactivation during cooling from 8 C observed by Johnson et al.

77 JOHNSON AND BUSTA 1 z <t 1 -j ~ -j CoOOLNG (1C /HR)- t n 46C 1 _ 1. ~ t 38 C * TOTAL POPULATON z 1.1-1 o. *9o 7 5 3 TEMPERATURE (C) FG. 1. Survival and germination of B. cereus F481/72 spores in TSB heated rapidly (ca. 9 centigrade degrees per h) to 9 C and cooled at 1 centigrade degrees per h. The total viable population () was initially ca. 16 spores per ml. Open symbols () represent spores resistant to 7 C for 15 min. (16a) was not the result of inactivation of sensitive spores during slow heating (2 or 4 C/h) to 8 C. nstead, inactivation of spores at temperatures between 46 and 38 C was stimulated by the higher heat treatment. Effect of constant temperature incubation in TSB on spore survival. Spores heated in TSB at 9 C for 2 min received approximately the same lethal heat treatment as spores being cooled from 9 to 8 C at 1 C/h (15). The response of heated spores to subsequent incubation at 35, 45, or 55 C is presented in Fig. 3. Significant inactivation was observed only with 45 C incubation. Maximal rates of inactivation occurred at 45 to 47 C (data not shown). Previous work has shown that germination of B. cereus spores does not occur at 55 C, slow germination occurs at 45 C, and optimum germination occurs at 35 C (16). ncubation at 45 C was selected for subsequent evaluation of the low-temperature spore inactivation. Previously reported D95 C-values for B. cereus strain F481/72 range from 5.6 (15) to 9.5 min (2), with a z-value of 8.9 C (15). From these data, one would not anticipate any measurable spore inactivation at 45 C (D45sC, ca. 16 min). Our results, however, show that spore inactivation can occur at 45 C with D-values of 96 to 12 min. Therefore, inactivation of this strain at 45 C seems to occur by a mechanism other than that involved in "normal" thermal inactivation of bacterial spores. a - APPL. ENVRON. MCROBOL. 1~~~~~~~~3 5 2 1 T N45 1 o 1 2 TME (HR) FG. 3. Survival of B. cereus F481/72 spores in TSB at 35 (), 45 (U), or 55 C (A) after initial heating in TSB at 9 C for 2 min. "nitial population" refers to the viable population after the 9 C heat treatment. Effect of germination on inactivation. Survival and germination of B. cereus spores in L-Ala and in buffer are presented in Fig. 4. Limited germination and no inactivation were apparent in buffer, whereas L-Ala supported significant initial germination and inactivation in 2 h. These data show that germination is required for 45 C inactivation to proceed. These data are also in agreement with the lack of spore inactivation at 55 C, a temperature above the germination temperature range (16). Effect of initial heat treatment on spore survival and germination at 45 C. Figure 5 illustrates the 45 C survival of B. cereus spores previously subjected to heat treatments at 7 to 9 C in TSB. The rate of inactivation of B. cereus spores in TSB at 45 C increased when the temperature of the initial heat treatment was increased. There was no appreciable lag before the onset of inactivation (P =.979). No inactivation occurred when spores were heated at 7 C. After initial heat treatments ranging from 7 to 9 C, the inactivation rate constant at 45 C can be determined by the following equation: logl(-slope) =.1135(7) - 12.24, where T is the temperature (degrees Celsius) of the initial heat treatment (r2 =.94). Figure 6 illustrates the effect of initial heat treatments on germination of spores at 45 C, calculated as the percentage of the viable population that was heat resistant at each sampling time. There is little difference in maximal germination among various initial heat treatments, with the exception of 9 C. This higher heat treatment may have caused a slightly lower level of germination. Severe heat treatments Z 2 o L-ALA BUFFER -J 14 o 6' Z 2O 6 3 TEMPERATURE ( C) FG. 2. Survival of B. cereus F481/72 spores during cooling at 1 centigrade degrees per h in TSB. noculated TSB was heated in boiling water to 8 (O) or 9 C () before cooling. 1 2 1 2 TME AT 45 C(HR) FG. 4. Germination and survival of heat-activated (7C for 15 min) B. cereus F481/72 spores at 45C in L-Ala and buffer. Closed symbols represent the total population, and open symbols represent the heat-resistant (7 C for 15 min) population of B. cereus.

VOL. 47, 1984 HEAT-NDUCED OUTGROWTH SENSTVTY N B. CEREUS 771 Z1 k-,v45 C..xiOl ~ O 1 9 - _i Z 1.~~~~~ P~ ~~~~ -.5 1 2 3 4 TME (HR) FG. 5. Survival of B. cereus F481/72 spores in TSB at 45 C after initial 15-min heat treatment at 7 (), 8 (O), 82.5 (A), 85 (O), 87.5 (V), or 9 C () and a 15-min holding period at 7 C. Solid lines represent data used in regression analysis. the spores apparently lacked this repair machinery. A previous report on the effect of cooling B. cereus spores in TSB and in a rice-beef extract medium from 9 C suggested that 45 C inactivation occurred in both media (16a). The apparent viable population increased during cooling from 35 to 1 C at a rate of 5 C/h in rice-beef extract; however, the population remained constant through this temperature range in TSB. There appears to be a component in rice that either protects spores from lethal damage or promotes growth at lower temperatures of the cooling process, suggesting that apparent 45 C inactivation may indeed be repairable. Effect of inhibition of macromolecular synthesis during germination and outgrowth of spores. The effect of inhibitors of protein, RNA, and DNA syntheses on outgrowth of B. cereus spores in TSB at optimum temperature (3 C) and at 45 C are presented in Fig. 8. At 3 C without inhibitors, B. cereus spores progressed rapidly from phase bright to phase dark, a majority of the spores were at the swollen stage by 1 (e.g., 9 C and perhaps 87.5 C) may have reduced the initial germination rate. These data demonstrate that the increased rate of 45 C inactivation observed with increased initial heat treatments was not due to the stimulation of more rapid germination. Response of spores in potential repair media. Figure 7 presents results representative of trials on the potential repair of spore damage induced by incubation in L-Ala at 45 C. Greater than 8% of the initial population was inactivated during 2 h of incubation at 45 C in L-Ala before the inoculation of recovery media with or without inhibitors of macromolecular synthesis. Little difference was noted among the responses for L-Ala with or without inhibitors (Fig. 7D). The same was true for starch (Fig. 7E). The increase of the popoulation in TSB, beef extract, and peptone was attributed to cell multiplication rather than spore repair since no increase in population was observed in the presence of any inhibitor. n these growth media, chloramphenicol, an inhibitor of protein synthesis, appeared to be less inhibitory than rifampin, nalidixic acid, and penicillin. Results for pyruvate, calcium lactate, magnesium sulfate, sucrose, glucose, and potassium phosphate at 35 C were similar to those of starch (data not shown). Rappaport and Goepfert (22) reported that injury of vegetative cells of B. cereus occurred at 47 C. Repair of this damage was possible in.1% peptone and required RNA synthesis. f this same type of damage occurred in our work, a 11 LO t Cc CD 11 z 5 a: C/) z -1 a- CLL a. 1 oi o x Z O w o a: U 8 ax 2 'k - \ 1 2 3 TME (HR) AT 45 C FG. 6. Percentage of germinated B. cereus F481/72 spores during 45 C incubation in TSB previously heated for 15 min at 7 (), 8 (O), 82.5 (A), 85 (O), 87.5 (V), or 9'C ( ), with holding at 7'C for an additional 15 min (see the text for details). - 4 TME (HR) FG. 7. Effect of 3'C incubation in potential repair media on B. cereus spores after inactivation at 45 C in L-Ala. Potential repair media included TSB (A),.1% beef extract (B),.1% peptone (C), L- Ala (D), and.1% soluble starch (E) with and without inhibitors of macromolecular synthesis. Symbols: L-Ala at 45'C (), no inhibitor (), nalidixic acid (O), chloramphenicol (A), rifampin (O), and penicillin (V).

772 JOHNSON AND BUSTA h, and elongation and division were apparent by 2.5 h. nhibition of RNA synthesis by rifampin had no effect on germination; however, swelling was limited and delayed. Furthermore, inactivation of <5% of the population occurred by 2.5 h. Swelling occurred normally in the presence of chloramphenicol, an inhibitor of the protein synthesis, and outgrowth did not proceed beyond this stage. The use of nalidixic acid, generally an inhibitor of DNA synthesis, delayed swelling, whereas elongated cells were observed after 2.5 h. By 4 h, many of these cells were extremely long and twisted, but no dividing cells were evident. Nalidixic acid did not inactivate outgrowing spores within 4 h at 3 C. When cells were incubated at 45 C without inhibitors, the change from phase bright to dark was slower than that observed at the optimum temperature (Fig. 8). Swelling and elongation were also delayed. No division was apparent in 4 h, and ca. 4% of the initial spore population was inactivated by this time. n the presence of rifampin, swelling was halted almost completely, and the population was reduced by 8% within 2.5 h. Chloramphenicol again halted outgrowth at the swelling stage, and inactivation of the population lagged behind the rate observed with inhibition of RNA synthesis. The response to nalidixic acid was similar to that for rifampin in both stage of outgrowth affected and inactivation of the population. These results agree with observations reported by others. Garrick-Silversmith and Torriani (7) reported that phase darkening of spores occurred before macromolecular synthesis. The synthesis of RNA has been shown to commence immediately after germination and peak during swelling (31). Swelling of spores is due primarily to the uptake of water and some nutrients (3); therefore, inhibition of RNA synthesis should result in primarily phase dark spores. nhibition of protein synthesis during outgrowth prevented cell wall synthesis (31); thus elongation would not occur. Temperaturesensitive mutants lacking protein synthesis were halted at the stage of swelling during outgrowth (6). Outgrowth proceeds in the absence of DNA replication C/) co a- C/) U- 11 1 5 55 E 2 cc 5OJflJ 9 Do 1 2 3 4 5 TME (HR) AT 3'C TME (HR) AT 45 C FG. 8. Effect of macromolecular synthesis inhibitors on B. cereus F481/72 spore survival ( ), germination, and outgrowth at 3 and 45 C in TSB. Spore suspensions were examined under phase contrast at 1,x and scored as phase bright (=), phase dark (_), swollen (i:i), elongated (Z), pairs (1), or ghost (171) spores. g, r rl-.- c U a 1A wi_ a)5- Cl), co C5- w APPL. ENVRON. MCROBOL. ~~~A t..~~~~~~.~ _c U 1 2 3 4 5 6 TME (HR) FG. 9. Effect of various initial heat treatments on B. cereus F481/72 survival ( ) and outgrowth at 45 C. (A) Spores heated at 75 C for 15 min before inoculation of TSB at 45 C; (B) spores in TSB heated at 8 C before incubation at 45 C; and (C) spores in TSB heated at 87.5 C before incubation at 45 C. Spores were examined under phase contrast at 1Ox and scored as phase bright (LO), phase dark (_), between dark and swollen (S). swollen (m), elongated (OW), or ghost (7) spores. (1). Ginsberg and Keynan (9) reported that mutants defective in DNA synthesis during outgrowth at 44 C formed long, curved cells with no division septa. The same morphology was observed with outgrowing parental strains incubated in "rich" media containing nalidixic acid. Hecker (13) reported that nalidixic acid may inhibit RNA synthesis in addition to DNA synthesis. n our studies, the synthesis of RNA, rather than DNA, may have been inhibited by nalidixic acid at nonpermissive temperatures, halting outgrowth at the phase dark or partially swollen stage. t appears that morphological characteristics of spores during outgrowth may be used as indicators of systems that are sensitive to inactivation treatments-a population of primarily phase dark spores resulting from inhibition of RNA synthesis, a majority of swollen spores indicating defective protein synthesis, and elongated cells suggesting interference with DNA replication. Stage of outgrowth sensitive to 45 C inactivation. Three heat activation treatments were employed to elicit differing levels of spore inactivation at 45 C. A 75 C heat treatment before inoculation of TSB at 45 C did not cause 45 C inactivation of B. cereus spores (Fig. 9A). Spores progressed normally through germination and outgrowth, and elongated spores were evident before 3 h. Heating of spores in TSB at 8 C resulted in a 5% reduction in the population at 45 C, and outgrowth appeared to be halted at swelling (Fig. 9B). Heating at 87.5 C before incubation at 45 C resulted in 45 C inactivation of 9% of the population, and outgrowth was halted at swelling (Fig. 9C). The cessation of outgrowth at the stage of swelling suggests that protein synthesis may be involved in inactivation of B. cereus F481/72 spores at 45C. The role of protein synthesis in 45 C inactivation of germinated B. cerelus spores is speculative. Three possible B

VOL. 47, 1984 HEAT-NDUCED OUTGROWTH SENSTVTY N B. CEREUS 773 functions should be considered: (i) incubation at 45 C may inhibit the synthesis of proteins that are required for survival of germinated spores at elevated temperature; (ii) heat activation before incubation at 45 C may inhibit the synthesis of proteins that are detrimental to the outgrowing spore; or (iii) protein synthesis is not altered, but the effects of an unrelated mechanism are expressed concurrently with protein synthesis. The second possibility is not consistent with data on the effect of chloramphenicol on spores incubated at 45 C (Fig. 8). nhibition of protein synthesis at 45 C resulted in a faster rate of inactivation than that occurring in TSB without inhibitor. Protein synthesis, therefore, appears to supply some protection of spores at 45 C. The spore proteins degraded during germination (25) appear to be similar to "heat shock" proteins (23). Both are associated with DNA (21, 27) and increase the melting point of DNA (25). Heat shock proteins are thought to protect DNA of bacteria, yeasts, and other species subjected to temperature up-shifts (4) and are produced by sporulating yeasts not previously exposed to heat (18). Bacterial spore proteins could serve the same function in B. cereus. Accelerated degradation of these proteins by activated proteases (29) would explain accelerated inactivation of germinated spores. This hypothesis is conjecture, and much work is required to verify these comments. Work in this area could also add information on the mechanism of heat resistance of bacterial spores. ACKNOWLEDGMENTS This work was funded by University of Minnesota Experiment Station project no. 18-59. We thank Lorraine B. Smith for her assistance with this work. LTERATURE CTED 1. Albertini, A. M., M. L. Baldi, E. Ferrari, E. snenghi, M. T. Zambelli, and A. Galizzi. 1979. Mutants of Bacillus subtilis affected in spore outgrowth. J. Gen. Microbiol. 11:351-363. 2. Berg, R. W., and W. E. Sandine. 197. Activation of bacterial spores. A review. J. Milk Food Technol. 33:435-441. 3. Busta, F. F. 1976. Practical implications of injured microorganisms in food. J. Milk Food Technol. 39:138-145. 4. Craig, E., T. ngolia, M. Slater, L. Manseau, and J. Bardwell. 1982. Drosophila, yeast and E. coli genes related to Drosophila heat-shock genes, p. 11-18. n M. J. Schlesinger, M. Ashburner, and A. Tissieres (ed.), Heat shock from bacteria to man. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 5. Galizzi, A., A. M. Albertini, M. L. Baldi, E. Ferrari, E. snenghi, and M. T. Zambelli. 1978. Genetic studies of spore germination and outgrowth in Bacillus subtilis, p. 15-157. n G. Chambliss and J. C. Vary (ed.), Spores V. American Society for Microbiology, Washington, D.C. 6. Galizzi, A., F. Gorrini, A. Rollier, and M. Polsinelli. 1973. Mutants of Bacillus subtilis temperature sensitive in the outgrowth phase of spore germination. J. Bacteriol. 113:1482-149. 7. Garrick-Silversmith, L., and A. Torriani. 1973. Macromolecular syntheses during germination and outgrowth of Bacillus subtilis spores. J. Bacteriol. 114:57-516. 8. Gilliland, S. E., F. F. Busta, J. J. Brinda, and J. E. Campbell. 1976. Aerobic plate count, p. 17-131. n M. L. Speck (ed.), Compendium of methods for the microbiological examination of foods, 1st ed. American Public Health Association, Washington, D.C. 9. Ginsberg, D., and A. Keynan. 1978. ndependence of Bacillus subtilis spore outgrowth from DNA synthesis. J. Bacteriol. 136:111-116. 1. Gottfried, M., C. Orrego, A. Keynan, and H. Halvorson. 1979. Specific inhibition of outgrowth of Bacillus subtilis spores by novobiocin. J. Bacteriol. 138:314-319. 11. Gould, G. W. 1969. Germination, p. 397-444. n G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press, nc., New York. 12. Gurney, T. R., and L. B. Quesnel. 198. Thermal activation and dry-heat inactivation of spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger. J. Appl. Bacteriol. 48:231-247. 13. Hecker, M. 1982. Effect of nalidixic acid on activation of RNA synthesis in outgrowing Bacillus subtilis spores. Z. Allg. Mikrobiol. 22:529-534. 14. Johnson, K. M., and F. F. Busta. 1983. Bacillus cereus spore response to static and dynamic temperature regimens, p. 74-75. n J. V. McLoughlin and B. M. McKenna (ed.), Research in food science and nutrition, vol. 2, Basic studies in food science. Boole Press, Dublin, reland. 15. Johnson, K. M., C. L. Nelson, and F. F. Busta. 1982. Germination and heat resistance of Bacillus cereus spores from strains associated with diarrheal and emetic food-borne illnesses. J. Food Sci. 47:1268-1271. 16. Johnson, K. M., C. L. Nelson, and F. F. 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