Planetary quarantine requirements associated with the launch of two Viking spacecraft necessitated microbiological assessment during assembly and testing at Cape Canaveral and the Kennedy Space Center. Samples were collected from selected surface of the Viking Lander Capsules (VLC), Orbiters, (VO), and Shrouds at predetermined intervals during assembly and testing. Approximately 7,000 samples were assayed. Levels of bacterial spores per square meter on the VLC-1 and VLC-2 were 1.6 x 10(2) and 9.7 x 10(1), respectively, prior to dry-heat sterilization. The ranges of aerobic mesophilic microorganisms detected on the VO-1 and VO-2 at various sampling events were 4.2 x 10(2) to 4.3 x 10(3) and 2.3 x 10(2) to 8.9 x 10(3)/m2, respectively. Approximately 1,300 colonies were picked from culture plates, identified, lypholipized, and stored for future reference. About 75% of all isolates were microorganisms considered indigenous to humans; the remaining isolates were associated with soil and dust in the environment. The percentage of microorganisms of human origin was consistent with results obtained with previous automated spacecraft but slightly lower than those observed for manned (Apollo) spacecraft.
Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 degrees C for 16 h and 24 degrees C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.
Bacillus subtilis var. niger spores were tested for dry-heat resistance on stainless-steel strips hung in an oven. Heat resistance was dependent on the relative humidity before and during treatment, which in turn affected the water content of the spores. Higher humidities increased the heat resistance of the spores. D-values ranged from 16.1 min for spores conditioned at <2% relative humidity (RH) and treated at 0.34% RH to 37.6 min for spores conditioned at 89% RH and treated at 1.1% RH. The y-intercept of the regression line ranged from 6.94 × 104 for spores conditioned and treated at the low humidities to 2.00 × 105 for spores conditioned at 89% RH and treated at 0.34% RH. For a constant value of N0, the y-intercept appears to be lowered by low-humidity conditions. The statistic log y0/log N0 is used to measure the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2% RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments.
Simulation of a heat process used in the terminal dry-heat decontamination of the Viking spacecraft is reported. Naturally occurring airborne bacterial spores were collected on Teflon ribbons in selected spacecraft assembly areas and subsequently subjected to dry heat. Thermal inactivation experiments were conducted at 105, 111.7, 120, 125, 130, and 135 degrees C with a moisture level of 1.2 mg of water per liter. Heat survivors were recovered at temperatures of 135 degrees C when a 30-h heating cycle was employed. Survivors were recovered from all cycles studied and randomly selected for identification. The naturally occurring spore population was reduced an average of 2.2 to 4.4 log cycles from 105 to 135 degrees C. Heating cycles of 5 and 15 h at temperature were compared with the standard 30-h cycle at 111.7, 120, and 125 degrees C. No significant differences in inactivation (alpha = 0.05) were observed between 111.7 and 120 degrees C. The 30-h cycle differs from the 5-and 15-h cycles at 125 degrees C. Thus, the heating cycle can be reduced if a small fraction (about 10-3 to 10-4) of very resistant spores can be tolerated.
The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (zD = 21 C), but an unusually low z for spores on paper (zD = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (zD = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (aw) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below aw 0.2 or above aw 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature.
Twenty-three soil samples were collected from areas of the United States where major spacecraft assembly and launch facilities are in operation. Soil samples were treated with ethyl alcohol, ultrasonic energy, and gross filtration. The resultant suspensions consisted of viable, naturally occurring bacterial spores and were used to inoculate stainless-steel strips. The strips were suspended in a forced air oven and assays were made at 5-min intervals for the number of viable spores. Most survivor curves were nonlinear. Subsequently, spore crops of heat-sensitive and heat-resistant soil isolates were found to have linear survivor curves at 125 C which were unaffected by the presence or absence of sterile soil particles from the parent sample. When two spore crops, one of which was heat-resistant and the other heat-sensitive, were mixed, the resultant nonlinear curves were unaffected by the presence or absence of sterile parent soil. Therefore, the survivor curves obtained originally with the soils were the result of heterogeneous spore populations rather than of protection afforded by soil particles in our test system. These results question the rationale both of assuming logarithmic death and of using decimal-reduction values obtained with subcultured standard reference spores in the derivation of dry-heat sterilization cycles for items contaminated with naturally occurring spore populations.
Inclusion of spores of Bacillus subtilis var. niger in water-soluble crystals increased the resistance of the spores to dry heat and to a gaseous mixture of methyl bromide and ethylene oxide. Resistance of spores in glycine crystals to dry heat at 125 C was increased 5 to 24 times compared to unprotected spores. There appeared to be a positive correlation between the size of the crystal and the degree of resistance. The resistance to dry heat of spores included in sodium chloride crystals was about six times greater than unprotected spores. A gaseous mixture of methyl bromide (964 mg/liter) and ethylene oxide (642 mg/liter) at 37% relative humidity was ineffective in sterilizing spores enclosed within these water-soluble crystals, as was ethylene oxide alone. However, if the relative humidity was sufficiently high to dissolve the crystals during exposure to the vapor, viable-spore counts were drastically reduced or were negative. The surfaces of crystals grossly contaminated with dry spores were sterilized by exposure to gaseous ethylene oxide. Sterilization of heat-labile or moisture-labile materials with a critical requirement for sterility, as in planetary probes or drugs, may be complicated by the presence of spores in naturally occurring water-soluble crystals. This phenomenon is similar to the protection afforded spores entrapped in solid plastics.
The dry-heat resistances of 70 bacterial spore isolates recovered from Mariner-Mars 1969 spacecraft were determined and expressed as D values (decimal reduction times). Fifty per cent of the spore isolates had D values of 60 min or less at 125 C. Of organisms with D values greater than 60 min, four were selected for a study of the effect of sporulation medium and suspension menstruum on dry-heat resistance. Both sporulation medium and suspension menstruum were found to affect significantly the dry-heat resistance of the bacterial spores tested.
The ability to determine the thermal resistance of naturally occurring air borne bacterial spores associated with spacecraft and their assembly areas has been hindered by lack of an effective collecting system. Efforts to collect and concentrate spores with air samplers or from air filters have not been successful. A fallout method was developed for this purpose and tested. Sterile Teflon ribbons (7.6 by 183 cm) were exposed in pertinent spacecraft assembly areas and subsequently treated with dry heat. Thermal inactivation experiments were conducted at 125 and 113 C. Heating intervals ranged from 1 to 12 h at 125 C and 6, 12, 18, and 24 h at 113 C. Eight hours was the longest heating time yielding survivors at 125 C, whereas survivors were recovered at all of the heating intervals at 113 C. D125C values were calculated using the fractional-replicate-unit-negative technique of Pflug and Schmidt (1968) and ranged from 25 to 126 min. This variation indicated that the most probable number of survivors at each heating interval did not fall on a straight line passing through the initial spore population. However, the most-probable-number values taken alone formed a straight line suggesting logarithmic thermal destruction of a subpopulation of spores with a D125C value of 6.3 h.
The currently used microbial decontamination method for spacecraft and components uses dry-heat microbial reduction at temperatures of >110°C for extended periods to prevent the contamination of extraplanetary destinations. This process is effective and reproducible, but it is also long and costly and precludes the use of heat-labile materials. The need for an alternative to dry-heat microbial reduction has been identified by space agencies. Investigations assessing the biological efficacy of two gaseous decontamination technologies, vapor hydrogen peroxide (Steris) and chlorine dioxide (ClorDiSys), were undertaken in a 20-m3 exposure chamber. Five spore-forming Bacillus spp. were exposed on stainless steel coupons to vaporized hydrogen peroxide and chlorine dioxide gas. Exposure for 20 min to vapor hydrogen peroxide resulted in 6- and 5-log reductions in the recovery of Bacillus atrophaeus and Geobacillus stearothermophilus, respectively. However, in comparison, chlorine dioxide required an exposure period of 60 min to reduce both B. atrophaeus and G. stearothermophilus by 5 logs. Of the three other Bacillus spp. tested, Bacillus thuringiensis proved the most resistant to hydrogen peroxide and chlorine dioxide with D values of 175.4 s and 6.6 h, respectively. Both low-temperature decontamination technologies proved effective at reducing the Bacillus spp. tested within the exposure ranges by over 5 logs, with the exception of B. thuringiensis, which was more resistant to both technologies. These results indicate that a review of the indicator organism choice and loading could provide a more appropriate and realistic challenge for the sterilization procedures used in the space industry.
A comparative study was made of the heat resistance of spores of putrefactive anaerobe 3679 grown in two different sporulation media and of the recovery pattern of these spores in several subculturing media after treatment with moist and dry heat. The heat resistance of the spores was characterized in the form of D and z values. The D values were determined by the modified Schmidt method. The z values were established by the graphic method. The results revealed significant differences in D and z values, depending on the type of heat and sporulation and subculture media. Spores grown in beef heart infusion showed higher heat resistance than those grown in Trypticase. Among the seven subculture media used, the largest number of spores was recovered in beef infusion. The magnitude of the D values at 121.1 C obtained with spores heated in moist heat decreased, depending on the subculture medium used, in the following order: beef infusion, pea infusion, yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. With spores subjected to dry heat, D values at 148.9 C decreased with the subculture medium in the following order: beef infusion, yeast extract, pea infusion and liver infusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained with spores subjected to dry heat were approximately double those obtained with moist heat. Their relative magnitude varied slightly, depending on the type of subculture medium used. However, the relative magnitudes of the D values and z values with reference to the subculture media used were different with moist heat from those obtained with dry heat. Two theories are discussed as possible explanations for the logarithmic order of death of bacterial spores. The results obtained in these experiments, together with the findings of other workers, are most compatible with the theory that heat treatment of spores results in an increased rate of random injury to the genetic material of the spores.
Heat-induced dormancy was observed when spores of two strains of Bacillus stearothermophilus were heated in distilled water at 80, 90, and 100 C. At temperatures above 100 C, true activation occurred; however, maximal activation was not achieved until temperatures of 110 to 115 C were employed. A heat treatment of 115 C for 3 min was required to induce maximal activation in one suspension of strain 1518 spores, whereas a heat treatment of 110 C for 7 to 10 min was adequate for the other suspension of strain 1518 spores. Spores from both strain M suspensions required heat treatments of 110 C for 9 to 15 min for maximal activation. The degree to which the spores could be activated was strain dependent and variable among spore suspensions of the same strain.
The germination and outgrowth of all spores, regardless of strain and suspensions source, were significantly reduced when the spores were heated in m/120 phosphate buffer at maximal or near maximal activating temperatures. It was suspected that phosphate lowered the heat resistance of the spores to the extent that the heat treatments were lethal to a portion of the populations.
Dry spores of Streptomyces viridochromogenes exhibited no endogenous metabolism when tested by the sensitive radiorespirometric technique. Wetting the spores resulted in a sharp increase in endogenous respiration followed by a gradual decrease to a constant level, whereby 0.02% of the spore carbon was respired to CO2 per h. The rate of endogenous metabolism increased slowly as unactivated and heat-activated spores germinated in a defined germination medium, reaching rapid rates only after germination was completed. Components of the defined germination medium, adenosine, l-alanine, and l-glutamic acid, were oxidized to CO2 at appreciable rates only after germination was complete. The QO2 (microliters of O2 uptake per hour per milligram [dry weight] of spores) values for endogenously respiring spores were 3.9 for unactivated and 7.8 for activated spores. Various sugars, amino acids, and organic acids were oxidized only slowly or not at all by unactivated and activated spores. The dry spores contained 5.2 × 10−2 μmol of ATP per g (dry weight). The ATP content of spores increased approximately 4-fold after suspension in buffer and approximately 11-fold after heat activation. During germination, the ATP level increased to a level of 1 μmol of ATP per g (dry weight) and remained constant. Germination was accompanied by excretion from the spores of approximately 8 and 12% of the total spore carbon from unactivated and activated spores, respectively. A potent germination inhibitor was released from the germinated spores. The germination inhibitor had no effect on heat-activated spores or spores which had begun germination for as short a time as 5 min.
Samples of soil collected from the Kennedy Space Center near the spacecraft assembly facilities were found to contain microorganisms very resistant to conventional sterilzation techniques. The inactivation kinetics of the naturally occurring spores in soil were investigated by using dry heat and ionizing radiation, first separately and then simultaneously. Dry-heat inactivation kinetics of spores was determined at 105 and 125 C; radiation inactivation kinetics was determined for dose rates of 660 and 76 krads/h at 25 C. Simultaneous combinations of heat and radiation were then investigated at 105, 110, 115, 120, and 125 C, with a dose rate of 76 krads/h. Combined treatment was found to be highly synergistic, requiring greatly reduced radiation doses to accomplish sterilization of the population.
Bacterial spore crops were prepared from 103 randomly selected aerobic mesophilic isolates collected during a spore assay of Mariner-Mars 1969 spacecraft conducted by the Jet Propulsion Laboratory. D125 c values, which were determined by the fractional-replicate-unit-negative-most-probable number assay method using a forced air oven, ranged from less than 5 min to a maximum of 58 min. Subsequent identification of the 103 isolates indicated that there was no relationship between species and dry-heat resistance. A theoretical dry-heat survival curve of the “population” was nonlinear. The slope of this curve was determined almost exclusively by the more resistant organisms, although they represented only a small portion of the “population.”
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.
Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (∼5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.
The heat resistance of dry bacterial spores was tested in various gases at temperatures ranging from 121.1 to 160 C (250 to 320 F). Spores of Clostridium sporogenes (PA 3679) were heated in air, carbon dioxide, and helium; spores of Bacillus subtilis 5230 were heated in these gases and also in oxygen and in nitrogen. The surrounding gas influenced the heat resistance, but the differences among gases were small. D values were about 7 min at 148.9 C (300 F); z values were about 18.3 C (33 F) for B. subtilis, and about 21.7 C (39 F) for C. sporogenes. The resistance of B. subtilis in carbon dioxide was about the same as in air, but lower than in all other gases; resistance in helium and nitrogen was about the same, and was higher than in all other gases. C. sporogenes had the least resistance in air; the resistance was about the same in carbon dioxide and helium. For B. subtilis, the gases in order of increasing heat resistance were carbon dioxide, air, oxygen, helium, and nitrogen, and for C. sporogenes, air, carbon dioxide, and helium. Neither oxygen content nor molecular weight of the gas appeared to have a marked influence on dry-heat resistance of the spores, whereas the more inert gases seemed to yield larger D values.
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.
Initiation of germination of heat-activated Streptomyces viridochromogenes spore occurs in media containing only calcium ions and organic buffer. The calcium-induced initiation of germination was accompanied by a decrease in absorbance of the spore suspension, an increased rate of endogenous metabolism, the loss of spore carbon, and the loss of heat resistance. Calcium amounts to 0.28% of the dry weight of freshly harvested spores. The amount of calcium remained the same after incubation of spores in water after heat activation. The spore content of calcium doubled after incubation in 0.5 mM CaCl2 for 5 min at 4 degrees C and during calcium-induced germination. Nearly all of the calcim appears to be bound to sites external to the spore membrane, since the chelating agents (ethylenedinitrilo) tetraacetic acid and arsenazo III removed virtually all of the calcium ions. The calcium ions must be present during the entire initiation of germination period. Germination ceases after an (ethylenedinitrilo) tetraacetic acid wash and begins again immediately after addition of calcium ions.
Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P2O5. Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.
A batch type heat pump assisted dehumidified air dryer was developed successfully with a medium range of temperatures (30–41°C) for safe drying of heat sensitive crops. Dehumidification system of the developed heat pump dryer (HPD) maintained the relative humidity (RH) of air entering the drying chamber below 40%. The inlet drying air temperature decreased during early hours of drying followed by rapid rise between the 2nd and 10th h, after which the temperature was almost stable. The RH of inlet and exhaust drying air increased initially and decreased subsequently with drying time as product became drier. The HPD was found to have a specific moisture extraction rate between 0.55 and 1.10 kg/kWh. Energy consumption for HPD for 24 h of operation was found less (4.48–5.05 kWh) than the hot air dryer (5.65–9.6 kWh) while operating under different drying conditions. Better quality dried sweet pepper (Capsicum annuum L.) was obtained in HPD owing to lower drying air temperature.
Heat pump dryer; Dehumidification system; Hot air dryer; Specific moisture extraction rate; Sweet pepper
Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water. Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90°C, the level of the 1:1 complex of Ca2+ and dipicolinic acid (CaDPA; ∼25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (Tlag), and then CaDPA was released within 1 to 2 min. (ii) The Tlag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all three species were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T1 when ∼80% of spore CaDPA was released, then increased rapidly until T2 when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T1. (vi) Carotenoids in B. megaterium spores' inner membranes exhibited two changes during heat treatment. First, the carotenoid's two Raman bands at 1,155 and 1,516 cm−1 decreased rapidly to a low value and to zero, respectively, well before Tlag, and then the residual 1,155-cm−1 band disappeared, in parallel with the rapid CaDPA release beginning at Tlag.
With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.
The thermal resistance characteristics of spores from strains of five different Bacillus species were determined in phosphate buffer and at relative humidities ranging from <0.001 to 100% in a closed-can system. Spores tested in the closed-can system showed a marked increase in heat resistance over those in phosphate buffer, with the greatest increases occurring at relative humidities between 1 and 50%. When estimates of the time to reduce the initial spore concentration 99.99% (F value) at eight different relative humidities were plotted against temperature, three different types of heat resistance profiles were obtained, with maximum resistances at relative humidities of 1, 7, and 30%. When the various strains of spores were heated at the relative humidity of their maximum heat resistance, their relative order of heat resistance was different from that seen in buffer. Spores from the soil isolate were most resistant under these conditions (F121.1 = 99.5 h).