Heat Resistance of Bacillus subtilis Spores at Various Water Activities

S ummary : The heat resistance of spores of Bacillus subtilis was determined in water vapour and in aqueous solutions of NaCl, LiCl, glucose and glycerol at various water activities. In water vapour and in glycerol solutions, maximal resistance appeared at low, but not zero water activity. In NaCl and glucose solutions only small variations in heat resistance occurred with decreasing water activity although a small increase was observed at the highest concentrations tested. With increasing concentrations of LiCl, heat resistance at first decreased but increased at water activities below 0·5. The possible interaction of compounds controlling water activity and water activity per se on heat resistance is discussed.     

Recovery of Airborne Streptococcal L-Forms at Various Relative Humidities

Aerosolized group A streptococcal L-forms survived better at low than at high relative humidities, and mid-range humidities were the most lethal. Colonial morphology of cells surviving aerosolization was not altered.     

Radiation Resistance of Spores of Bacillus subtilis and B. stearothermophilus at Various Water Activities

S ummary . The radiation resistance of spores of Bacillus subtilis and B. stearothermophilus was determined under anoxic conditions in water vapour and in aqueous solutions of glucose and glycerol at various water activities. In water vapour, resistance of both kinds of spores increased slightly with decreasing water activity ( a _w). In glycerol solutions, a phase of rapid increase in resistance with decreasing a _w was followed by a slower increase on further reduction of a _w. This second phase was almost parallel to the water vapour curve, which, however, showed considerably lower resistance. The initial phase of rapid increase in resistance was also observed in glucose solutions.     

Heat Resistance and Population Stability of Lyophilized Bacillus subtilis Spores

Bacillus subtilis 5230 spores were lyophilized in 0.067 M phosphate buffer and stored at 2 to 8°C for 9 to 27 months. The lyophilized spores were reconstituted with buffer or 0.9% saline, and the heat resistance was determined in a thermoresistometer. Lyophilization had no effect on the heat resistance of the spores but did result in a slight decrease in population (≤0.3-logarithm reduction). The lyophilized spores maintained heat resistance and population levels over the test periods. The D-values ranged from 0.44 to 0.54 min at 121.1°C, and the z-values ranged from 6.1 to 6.6°C. Lyophilization was concluded to be an acceptable alternative for storage of bacterial spores that are to be used as biological indicators in sterilization processes.     

Heat Shock Affects Permeability and Resistance of Bacillus stearothermophilus Spores

[This corrects the article on p. 2517 in vol. 54.].     

Heat Resistance Correlated with DNA Content in Bacillus megaterium Spores

Two subpopulations of Bacillus megaterium spores (1.360 and 1.355 g/ml) were obtained by density gradient centrifugation. The heavier spores had a higher thermoresistance (e.g., D₈₀ = 186 versus 81 min) and a higher DNA content (1.25 × 10⁻¹⁴ versus 0.65 × 10⁻¹⁴ g per spore, apparently corresponding to digenomic versus monogenomic spores). No appreciable differences were found in the mineral and dipicolinic acid contents or in the inactivation kinetics of the two subpopulations. The implications of the findings are discussed with regard to mechanisms of heat resistance and of inactivation.     

Heat Injury of Bacillus subtilis Spores at Ultrahigh Temperatures

The following three criteria indicated that Bacillus subtilis A spores were injured, but not completely inactivated, by ultrahigh temperature treatment. (i) Significant reductions in survivors were observed when spores were enumerated with a standard medium but not when the medium contained added CaCl(2) and sodium dipicolinate. (ii) After a damaging heat treatment, more survivors were enumerated with the standard medium after incubation at 32 C than at 45 C, which was opposite to the result with untreated or slightly heated spores. (iii) Apparent numbers of survivors increased during the initial period of 3 C storage when enumerated with the standard medium at 45 C. No injury was evident when survivors were enumerated at either incubation temperature with the medium containing added CaCl(2) and sodium dipicolinate. Heat activation of the spores did not significantly influence the appearance of heat injury. The data suggested that the heat injury occurred in a germination system which was required in the absence of CaCl(2) and sodium dipicolinate.     

Effect of Ultrasonic Waves on the Heat Resistance of Bacillus cereus and Bacillus licheniformis Spores

Heat resistance of Bacillus cereus and Bacillus licheniformis spores in quarter-strength Ringer solution decreases markedly after ultrasonic treatments which are unable to kill a significant proportion of the spore population. This effect does not seem to be caused by a loss of Ca(2+) or dipicolinic acid. The use of ultrasonics to eliminate vegetative cells or to break aggregates in Bacillus spore suspensions to be used subsequently in heat resistance experiments appears to be unadvisable.     

Sporulation and Heat Resistance of Bacillus stearothermophilus Spores Produced in Chemically Defined Media

The effect of amino acids on sporulation is discussed. Heat-resistant spores were produced in a chemically defined medium.     

Effects of Minerals on Resistance of Bacillus subtilis Spores to Heat and Hydrostatic Pressure

Among Bacillus subtilis IFO13722 spores sporulated at 30, 37, and 44°C, those sporulated at 30°C had the highest resistance to treatments with high hydrostatic pressure (100 to 300 MPa, 55°C, 30 min). Pressure resistance increased after demineralization of the spores and decreased after remineralization of the spores with Ca²⁺ or Mg²⁺, whereas the resistance did not change when spores were remineralized with Mn²⁺ or K⁺, suggesting that former two divalent ions were involved in the activation of cortex-lytic enzymes during germination.     

Dry Heat Inactivation of Bacillus subtilis var. niger Spores as a Function of Relative Humidity

Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations.     

Resistance and Structure of Spores of Bacillus subtilis

Spores of Bacillus subtilis NCDO 2130 were produced on five different media and their structure and resistance to chemicals, to heat and to ultraviolet (uv) irradiation compared. Resistance to one chemical was not necessarily related to resistance to another, to uv irradiation or to heat. Spores with the smallest protoplasts and largest cortices were most resistant to heat, while the least resistant were those in which the protoplasts became electron dense after staining and which had the lowest concentrations of calcium and dipicolinic acid.     

Resistance of Bacillus Spores to Combined Sporicidal Treatments

S ummary . Moist heat at 82° (100° for Bacillus stearothermophilus ) and solutions of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride at 24° separately showed no sporicidal activity against B. pumilis, B. stearothermophilus, B. subtilis and B. subtilis var. niger . Spores of the last organism were the most sensitive to γ radiation, the D value being 0.16 Mrad. Prior irradiation with a dose of 0.16 Mrad brought about only a slight increase in the sensitivity of the spores to moist heat. The presence of bactericide during irradiation did not affect radiation resistance. Inactivation rates were greater when the spores were heated in the presence of a bactericide than in aqueous suspension and benzalkonium chloride was more active than chlorocresol. Chlorocresol enhanced the heat activation of B. stearothermophilus at 100°. Irradiation in the presence of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride had no effect on the subsequent resistance of the spores when heated in the presence of these bactericides. It is concluded that it is unlikely that combinations of moist heat, radiation and bactericides, each less severe than when used in an accepted sterilization process, will lead to an alternative process which, while less damaging to the materials being sterilized, would still maintain the accepted standards of freedom from contamination.     

Rhythmic Changes in Dry Heat Resistance of Bacillus subtilis Spores After Rapid Changes in pH

Heat resistance of freeze-dried Bacillus subtilis spores varied in a rhythmic manner as a function of time between acidification to about pH 1.5 and freezing. A comparable rapid shift to pH 11 produced little change in resistance to heat.     

Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.Spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of stress factors (31, 32). While spores can remain dormant for long periods, if given the proper stimulus, they can rapidly “return to life” in the process of spore germination followed by outgrowth (30). Since spores are generally present in significant amounts on many foodstuffs and growing cells of a number of Bacillus species are significant agents of food spoilage and food-borne disease (32), there is continued applied interest in spore resistance and germination. While dormant spores can be killed by a treatment such as wet heat, this requires high temperatures that are costly and detrimental to food quality. Consequently, there has long been interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. However, this strategy has been difficult to apply because of the significant heterogeneity in germination rates between individual spores in populations. One reflection of this heterogeneity is the extremely variable lag times following addition of germinants but prior to initiation of germination events; while these lag times can vary from 10 to 30 min for most spores in populations, some spores have lag times of many hours or even many days (2, 12, 13, 15, 25). The spores that are extremely slow to germinate have been termed superdormant spores, and populations of superdormant spores have recently been isolated from three Bacillus species, and their germination properties characterized (9, 10). These superdormant spores germinate extremely poorly with the original germinants used to remove dormant spores from spore populations, thus allowing superdormant spore isolation, and also poorly with a number of other germinants, in particular, germinants that target nutrient germinant receptors different than those activated to isolate the superdormant spores. However, the superdormant spores germinate reasonably well with mixtures of nutrient germinants that target multiple germinant receptors. All reasons for spore superdormancy are not known, but one contributing factor is the number of nutrient germinant receptors in the spore''s inner membrane that trigger spore germination by binding to nutrient germinants (9). The levels of these receptors are most likely in the tens of molecules per spore (24), and thus stochastic variation in receptor numbers might result in some spores with such low receptor numbers that these spores germinate very poorly (23). Indeed, 20- to 200-fold elevated levels of at least one nutrient germinant receptor greatly decreases yields of superdormant spores of Bacillus subtilis (9).Spores of Bacillus species generally exhibit a requirement for an activation step in order to exhibit maximum germination (17). Usually this activation is a sublethal heat treatment that for a spore population exhibits an optimum of 60 to 100°C depending on the species. Spores are also extremely resistant to wet heat, generally requiring temperatures of 80 to 110°C to achieve rapid spore killing, with the major factor influencing the wet-heat resistance of spores of mesophilic strains being the spore core''s water content, which can be as low as 30% of wet weight as water in a fully hydrated spore (8, 19, 27, 28, 31). Invariably, increases in core water content are associated with a decrease in spore wet-heat resistance (8, 19, 22, 25). While spore populations most often exhibit log-linear kinetics of wet-heat killing, the observation of tailing in such killing curves at high levels of killing is not uncommon, suggesting there is significant heterogeneity in the wet-heat resistances of individual spores in populations (27, 28). While there has been no comparable work suggesting that there is also heterogeneity in the temperature optima for heat activation of individual spores in populations, this certainly seems possible and indeed was suggested as one cause of spore superdormancy, as yields of superdormant spores from spore populations that are not heat activated are much higher (9, 10). Consequently, the current work was initiated to test the hypothesis that superdormant spores require heat activation temperatures that are higher than those of the original dormant spores. Once this was found to be the case, the wet-heat resistance and core water content of the superdormant and original dormant spores were compared, and the environment of the spore core''s major small molecule, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) was assessed by Raman spectroscopy of individual spores.     

The Effect of Sodium Chloride on Heat Resistance and Recovery of Heated Spores of Bacillus stearothermophilus

Increasing concentrations (2, 4 and 8% w/v) of sodium chloride in the heating medium progressively reduced the heat resistance of spores of Bacillus stearothermophilus. Storage at 4° in water or in sodium chloride solutions had little effect on viable counts of unheated spores, but with the increase in sodium chloride concentration there was a reduction in the heat activation effect and a small decrease in heat resistance of the spores. Increasing the severity of heat treatment rendered spores increasingly sensitive to sodium chloride in the plating medium.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Role of Disulphide Bonds in the Resistance of Bacillus cereus Spores to Gamma Irradiation and Heat

S ummary . Spores of Bacillus cereus were treated with thioglycollic acid which ruptures at least 10–30% of the spore disulphide bonds by reducing them to thiol groups. The treated spores were still viable and were sensitive to lysozyme but remained as resistant to γ-irradiation and to heat as untreated spores. Neither treated nor untreated spores were sensitized to irradiation by reagents which block thiol groups. The results did not indicate that the high content of disulphide bonds in spore coat protein protects spores against inactivation by irradiation or heat.     

Heat Activation and Heat-induced Dormancy of Bacillus stearothermophilus 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.