Use of a computer data file for storage of heat resistance data on bacterial spores

The use of a computer data file to compile published heat resistance data on bacterial spores is described. Information relating to sporulation, heating and recovery of the spores, in addition to decimal reduction times at different temperatures has been stored. The data file was constructed using Minitab and an indexer program on a VAX 11/750 computer operating on VMS. Storage of the data in this way enables rapid manipulation and analysis of large quantities of information relating to heat resistance of bacterial spores.     

Mineralization and heat resistance of bacterial spores.

The heat resistances of the fully demineralized H-form spores of Bacillus megaterium ATCC 19213, B. subtilis var. niger, and B. stearothermophilus ATCC 7953 were compared with those of vegetative cells and native spores to assess the components of resistance due to the mineral-free spore state, presumably mainly from dehydration of the spore core, and to mineralization. Mineralization greatly increased heat resistance at lower killing temperatures but appeared to have much less effect at higher ones.     

Effect of lipid materials on heat resistance of bacterial spores

The apparent heat resistance of spores of Bacillus megaterium, B. subtilis, B. cereus, B. stearothermophilus, and Clostridium botulinum type E in lipids was investigated and compared with the resistance of the spores in phosphate buffer solution. The most pronounced increase in heat resistance was noted for B. subtilis and C. botulinum type E, the increase varying with the type of lipid used. A high water content of the lipids used as heating menstruum lowered the heat resistance of the spores. Possible explanations for the high heat resistance of spores in lipids are discussed.     

Dehydration partitioned within core protoplast accounts for heat resistance of bacterial spores

Abstract In Escherichia coli , adenosine 3',5'-cyclic monophosphate (cAMP) is excreted into the growth media. Making use of a phosphodiesterase as scavenger of extracellular cAMP we show that: (i) extracellular cAMP does not interfere with cellular functions; (ii) transient accumulation of cAMP, followed by its rapid excretion, elicits a severe repression of catabolic enzymes.     

Protoplast dehydration correlated with heat resistance of bacterial spores.

Water content of the protoplast in situ within the fully hydrated dormant bacterial spore was quantified by use of a spore in which the complex of coat and outer (pericortex) membrane was genetically defective or chemically removed, as evidenced by susceptibility of the cortex to lysozyme and by permeability of the periprotoplast integument to glucose. Water content was determined by equilibrium permeability measurement with 3H-labeled water (confirmed by gravimetric measurement) for the entire spore, with 14C-labeled glucose for the integument outside the inner (pericytoplasm) membrane, and by the difference for the protoplast. The method was applied to lysozyme-sensitive spores of Bacillus stearothermophilus, B. subtilis, B. cereus, B. thuringiensis, and B. megaterium (four types). Comparable lysozyme-resistant spores, in which the outer membrane functioned as the primary permeability barrier to glucose, were employed as controls. Heat resistances were expressed as D100 values. Protoplast water content of the lysozyme-sensitive spore types correlated with heat resistance exponentially in two distinct clusters, with the four B. megaterium types in one alignment, and with the four other species types in another. Protoplast water contents of the B. megaterium spore types were sufficiently low (26 to 29%, based on wet protoplast weight) to account almost entirely for their lesser heat resistance. Corresponding values of the other species types were similar or higher (30 to 55%), indicating that these spores depended on factors additional to protoplast dehydration for their much greater heat resistance.     

The source of the heat resistance of bacterial spores: Study of water in spores by NMR

It has been postulated that the heat stabilization of the essential macromolecules in the core of the spore may be produced by dehydration at two levels: (i) the spore is drier at high relative humidity than the vegetative cell and (ii) the core of the spore may be less hydrated than the cortex and the coat. The latter postulate was subjected to experimental testing by ¹H-NMR studies of the water signal in the five species of spores and coat and (coat + cortex) preparations. The transverse relaxation rate $\text{(}\text{1}\text{T}{}_2\text{)}$ was determined in samples equilibrated at constant relative humidity. To allow for the effect of paramagnetic ions on $\text{1}\text{T}{}_2$ a model system (wool keratin) was studied in the presence of known amounts of Ca(II), Mn(II), Cu(II), Ni(II) and Fe(III). Because of the dominant effect of Mn(II) on $\text{1}\text{T}{}_2$, the effect of small amounts of other metal ions in spores was neglected. The relaxation rate of water at a particular relative humidity and manganese concentration was consistently less for intact spores than for coat or coat + cortex, hence the water in the core is more mobile than in the outer integuments. Sorption isotherm studies have shown that at a particular relative humidity there is about as much water in the core as in the cortex and coat. These two results taken together indicate that the hypothesis that the core is drier than the cortex and coat is incorrect, hence the spore is not heat-stabilized in this way. A theory is proposed in which heat stabilization is attributed to immobilization of essential enzymes and nuclei acids by a solid support, calcium dipicolinate, in a similar fashion to the heat stabilization of enzymes in a charged polymer matrix. It is proposed that stabilization is effected by electrostatic and hydrogen bonds between the calcium dipicolinate and the essential macromolecules. Experiments in progress show that enzymes and DNA are heat-stabilized in vitro by calcium dipicolinate.     

Perturbation of the heat resistance of bacterial spores by sporulation temperature and ethanol

Spores ofBacillus megaterium, B. subtilis, andB. stearothermophilus, harvested from cultures grown and sporulated at different temperatures or in the presence of ethanol, had different thermal resistance. There was a direct relationship between the sporulation temperature and the spore-killing temperature. The spores were more temperature-sensitive when formed in ethanol-supplemented media. Temperature and ethanol are known to perturb the degree of order within membranes and to alter membrane functions. Thus, alteration of spore membranes is an additional factor in the multifactorial nature of heat resistance. Another interpretation may be that heat shock proteins, known to be induced by heat, are formed during sporulation and may increase the thermostability of the spores.     

Effect of the internal water activity of bacterial spores on their heat resistance in water

The water contents and effective a_w of the core, cortex, and coat of spores in water, as well as the masses of the core, cortex, and coat plus exosporium in the dry state, are calculated from volumes, dry densities, and water absorption isotherms of the sporal components. From data presented here for spores ofBacillus subtilis var.niger andBacillus cereus T, and from previously published data forBacillus stearothermophilus, the logarithm of the heat resistance of the spores in water is linearly related to the effective a_w of their core and cortex.     

The heat resistance of bacterial spores due to their partial dehydration by reverse osmosis

The ability of bacterial spores to withstand heat is known to be associated with a lowering of their water content. This partial dehydration is considered to be produced by reverse osmosis, with the pressure being applied by the cortex as it is growing. Experiments show that the cortex is capable of supplying the pressure.     

Study of heat resistance in a bacterial nucleosidase

The enzyme nucleosidase (EC. 3.2.2.1.) is present in the intact spores, germinated spores as well as vegetative cells of $\text{Bacillus cereus}$ T. In the intact spores the enzymeis resistant to heat and, in fact, has a high temperature optimum. Though the spores themselvesbecome sensitive to heat on germination, the enzyme retains its resistance to heat on germination as well as its high temperature optimum. The vegetative cell enzyme is sensitive to heat. The enzyme in all types of cells &; spores is resistant to octyl alcohol. There is a close correlation between the development of heat resistance in the sporulating cells and that of heat resistance of the enzyme.     

Thermal resistance of naturally occurring airborne bacterial spores.

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.