Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.     

Survival and Outgrowth of Clostridium botulinum Type E Spores in Smoked Fish

Chub injected in the loin muscle with 10(6)Clostridium botulinum type E spores were smoked to an internal temperature of 180 F (82.2 C) for 30 min, sealed in plastic bags, and incubated at room temperature (20 to 25 C) for 7 days. Viable type E spores were found in practically all such fish. Toxin formation by the survivors in the smoked fish was dependent on the brine concentration of the smoked fish. A brine concentration of 3% or higher, as measured in the loin muscle, inhibited toxin formation. Six different type E strains gave similar results. Only a few hundred of the million spores in the inoculum survived the smoking. Moisture in the atmosphere during smoking did not reduce the incidence of fish with type E survivors.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Influence of NaCl, NaNO2 and oxygen on the germination and growth of Bacillus licheniformis, a spoilage organism of chub-packed luncheon meat

The thermal resistance of Bacillus licheniformis spores was increased from a D70-value of 590 min to one of 900 min by the addition of 4% NaCl to the heating medium [tryptone-yeast extract-glucose (TYG) broth, pH 6.8], but was decreased to 470 min in TYG broth acidified to pH 4.4. Sodium nitrite (0.02%) enhanced spore destruction at 80 degrees C but not at 70 degrees C; addition of 4% NaCl eliminated this effect. Less than half the number of spores surviving heat comparable to commercial cooking were heat-damaged to the extent of being unable to grow aerobically in the presence of 4% NaCl. No growth occurred during anaerobic incubation even when the media contained no added NaCl. Oxygen was not required to trigger spore germination, but trace amounts were needed for the successful outgrowth of germinated spores. Spore germination was accelerated and enhanced by the presence of at least 2% NaCl. Therefore under anaerobic conditions NaCl promotes microbiological stability because the germinated spores cannot develop further and become moribund. It is concluded that the plastic casing of luncheon-meat chubs is not sufficiently oxygen-impermeable to allow the product a long shelf-life other than at chill temperatures unless the chubs are stored in an oxygen-free atmosphere.     

Fungal and actinomycete spore aerosols measured at different humidities with an aerodynamic particle sizer

T.M. MADELIN AND H.E. JOHNSON. 1992. An aerodynamic particle sizer (APS) that uses laser Doppler velocimetry was used to determine aerodynamic diameters of spores of fungal and thermophilic actinomycete species common in mouldy hay, acrosolized at different humidities and temperatures. Results were compared with those obtained from inertial impaction in a cascade impactor. The APS gave slightly smaller measurements than the cascade impactor. Both methods gave aerodynamic diameters generally slightly smaller than the average spore dimensions observed on cascade impactor slides with a microscope. The latter measurements were less than axial dimensions given in the literature. Brief passage of spores through air at 95% relative humidity (RH) and 38°C, compared with 40% RH and 20°C, caused an immediate increase in their aerodynamic diameter and the breaking of chains of spores. Cultures maintained at 75% RH and aerosolized at 98% RH similarly produced larger spore particles than those passed through dry air. These findings have implications for mould-induced asthma and allergic alveolitis since they relate to physical behaviour of airborne spores and particle deposition sites in the lung.     

Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores

Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0 °C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable, temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1% in 14 days.     

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Influence of NaCl, NaNO2 and oxygen on the germination and growth of Bacillus licheniformis, a spoilage organism of chub-packed luncheon meat

The thermal resistance of Bacillus licheniformis spores was increased from a D ₇₀-value of 590 min to one of 900 min by the addition of 4% NaCl to the heating medium [tryptone-yeast extract-glucose (TYG) broth, pH 6.8], but was decreased to 470 min in TYG broth acidified to pH 4.4. Sodium nitrite (0.02%) enhanced spore destruction at 80°C but not at 70°C; addition of 4% NaCl eliminated this effect. Less than half the number of spores surviving heat comparable to commercial cooking were heat-damaged to the extent of being unable to grow aerobically in the presence of 4% NaCl. No growth occurred during anaerobic incubation even when the media contained no added NaCl. Oxygen was not required to trigger spore germination, but trace amounts were needed for the successful outgrowth of germinated spores. Spore germination was accelerated and enhanced by the presence of at least 2% NaCl. Therefore under anaerobic conditions NaCl promotes microbiological stability because the germinated spores cannot develop further and become moribund. It is concluded that the plastic casing of luncheon-meat chubs is not sufficiently oxygen-impermeable to allow the product a long shelf-life other than at chill temperatures unless the chubs are stored in an oxygen-free atmosphere.     

Effect of storage time and temperature and the variation among replicate tests (on different days) on the performance of spore disks and strips.

Laboratory-prepared spore disks were stored for 96 weeks at 22 degrees C with 50% relative humidity (RH) and at 4 degrees C with less than 1% RH. At the same time commercial spore strips were stored for 64 weeks at 22 degrees C with 50% RH. The spore count per unit and the heat resistance were measured at the beginning of the experiment and after 16, 32, 48, 64, 80, and 96 weeks of storage. The laboratory-prepared spore disks stored at 4 degrees C with less than 1% RH showed less change in numbers of spores per disks and decrease in the survival time than did the disks stored at 22 degrees C with 50% RH. Both the laboratory-prepared spore disks and the commercial spore strips stored at 22 degrees C with 50% RH decreased in survival times with increased storage time. The relative change in the survival times with storage was less for the commercial spore strips than for the laboratory-prepared spore disks.     

Sensitivity of an Enrichment Culture Procedure for Detection of Clostridium botulinum Type E in Raw and Smoked Whitefish Chubs

The sensitivity of an enrichment culture procedure for detecting Clostridium botulinum type E in whitefish chubs (Leucichthys sp.) was assayed. Data demonstrated that fish inoculated with 10 or more viable C. botulinum spores regularly develop specifically neutralizable enrichment cultures. Mild heat treatment (60 C, 15 min) substantially reduced the sensitivity of enrichment culturing. This effect was particularly noticeable in the culturing of fish which harbored fewer than 10 spores each. Evidence is presented which indicates that sensitivity of enrichment, without heat, approaches the level of one spore per fish. Smoked whitefish chubs, containing from one to several hundred spores each, were examined for toxin content after storage at 5, 10, 15, and 28 C for as long as 32 days. The lowest temperature at which detectable toxin was produced was 15 C. This occurred in 1 of 10 fish incubated for 14 days. C. botulinum was regularly recovered, by enrichment culture, from fish inoculated with small numbers of spores, even though toxin was not detected by direct extraction of incubated fish. Persistence of C. botulinum type E spores was observed to decline with an increase in the temperature and time at which inoculated fish were stored.     

Fungal and actinomycete spore aerosols measured at different humidities with an aerodynamic particle sizer.

An aerodynamic particle sizer (APS) that uses laser Doppler velocimetry was used to determine aerodynamic diameters of spores of fungal and thermophilic actinomycete species common in mouldy hay, aerosolized at different humidities and temperatures. Results were compared with those obtained from inertial impaction in a cascade impactor. The APS gave slightly smaller measurements than the cascade impactor. Both methods gave aerodynamic diameters generally slightly smaller than the average spore dimensions observed on cascade impactor slides with a microscope. The latter measurements were less than axial dimensions given in the literature. Brief passage of spores through air at 95% relative humidity (RH) and 38 degrees C, compared with 40% RH and 20 degrees C, caused an immediate increase in their aerodynamic diameter and the breaking of chains of spores. Cultures maintained at 75% RH and aerosolized at 98% RH similarly produced larger spore particles than those passed through dry air. These findings have implications for mould-induced asthma and allergic alveolitis since they relate to physical behaviour of airborne spores and particle deposition sites in the lung.     

Purification and characterization of additional low-molecular-weight basic proteins degraded during germination of Bacillus megaterium spores.

Dormant spores Bacillus megaterium contained a group of low-molecular-weight (5,000 to 11,000) basic (pI greater than 9.4) proteins (termed D, E, F, and G proteins) which could be extracted from disrupted spores with strong acids. These proteins were distinct from the previously described A, B, and C proteins which are degraded during spore germination. However, the D, E, F, and G proteins were also rapidly degraded during spore germination, accounting for 10 to 15% of the protein degraded. Proteins similar to the D, E, F, and G species were also present in spores of other bacterial species. In B. megaterium, the D, E, F, and G proteins were low or absent (less than 15% of the spore level) in vegetative and young sporulating cells and appeared only late in sporulation. The D, E, F, and G proteins were purified to homogeneity, and all contained a high percentage of hydrophilic amino acids; one protein (G) contained 31% basic amino acids and also contained tryptophan. All four proteins were rapidly degraded in vitro by dormant spore extracts. Two proteins (D and F) were degraded in vitro by the previously described spore protease which initiates degradation of the A, B, and C proteins in vivo; the spore enzyme (s) degrading proteins E and G have not been identified.     

Contribution of Spores to the Ability of Clostridium difficile To Adhere to Surfaces

Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student''s t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species.     

Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum

Heat treatment of spores of non-proteolytic Clostridium botulinum at 85°C for 120 min followed by enumeration of survivors on a medium containing lysozyme resulted in a 4.1 and 4.8 decimal reduction in numbers of spores of strains 17B (type B) and Beluga (type E), respectively. Only a small proportion of heated spores formed colonies on medium containing lysozyme; this proportion could be increased by treatments designed to increase the permeability of heated spores. The results indicate that the germination system in spores of non-proteolytic Cl. botulinum was destroyed by heating, that lysozyme could replace this germination system, and that treatments that increased the permeability of the spore coat could increase the proportion of heated spores that germinated on medium containing lysozyme. These results are important in relation to the assessment of heat-treatments required to reduce the risk of survival and growth of non-proteolytic Clostridium botulinum in processed (pasteurized) refrigerated foods for extended storage.     

Effect of Sodium Chloride and pH on the Outgrowth of Spores of Type E Clostridium botulinum at Optimal and Suboptimal Temperatures

The sodium chloride inhibition of spore outgrowth of four strains of type E Clostridium bolulinum was determined in a Trypticase-peptone-glucose (TPG) medium. At 16, 21, and 30 C, spores of three strains required 5.0% and one strain 4.5% salt for complete inhibition during 1 year of incubation. At 8 and 10 C, spores of the four strains required 4.5% salt for definite inhibition. Salt concentrations slightly lower than those providing inhibition tended to extend spore outgrowth time at low temperatures. The minimal pH permitting outgrowth of type E spore inocula was affected by the concentration of reducing compound present in the system. When either 0.02% sodium thioglycolate or 0.05% L-cysteine hydrochloride was used, outgrowth at 30 and 8 C occurred at much lower pH levels than when 0.2% thioglycolate was added. At 30 C, spores of one strain showed outgrowth in TPG medium as low as pH 5.21 with an inoculum of 2 million spores per replicate tube. At a 10-fold higher inoculum, the same strain showed outgrowth at pH 5.03 in one of five replicate tubes. At 8 C, spore outgrowth of the four strains occurred at pH 5.9, but not at pH 5.7, in TPG medium containing L-cysteine hydrochloride.     

Thermal inactivation and injury of Bacillus stearothermophilus spores.

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Thermal inactivation and injury of Bacillus stearothermophilus spores

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Heat stability of Bacillus cereus enzymes within spores and in extracts.

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.