Studies on the mechanism of action of ionizing radiations. VII. Cellular respiration,cell division,and ionizing radiations

On x-irradiation of the eggs and sperm of Arbacia punctulata there was inhibition of respiration with relatively large doses, whereas there was an increase with small doses. The dose required to produce an increase of respiration depended on the degree of sensitivity of the cell to the effect of ionizing radiation. Sperm cells were more sensitive; then came fertilized eggs; unfertilized eggs were the least sensitive. The inhibiting effect of x-rays on cell division was observed even on irradiation with x-ray doses which produced an increase of respiration. These results are compared to similar effects produced by thiol reagents and are attributed to oxidation of the thiol compounds in the cell.     

Role of the coat in resistance of bacterial spores to inactivation by ethylene oxide

A study of spore morphology revealed an association between resistance to ethylene oxide and abnormal spore coat development and both characteristics were enhanced by selection and propagation of resistant survivors. After rupture of the coat, spores were sensitized to lysozyme but not to ethylene oxide. Resistance to ethylene oxide was increased following treatment of spores with ureamercaptoethanol-alkali and decreased after treatment with urea. Germinated spores retained resistance to the sterilant and loss of resistance coincided with emergence of the vegetative cells.     

Method to sensitize bacterial spores to subsequent killing by dry heat or ultraviolet irradiation

Hydrogen peroxide and ultraviolet irradiation are known to interact synergistically for killing of bacterial spores. Synergy could be demonstrated with spores of Bacillus megaterium ATCC19213 adsorbed to filter paper strips or glass coverslips treated first with the peroxide and then dried for as long as 48 h prior to UV irradiation. This delayed action was considered to be due to absorption of the peroxide by the spores in an active but not readily vaporized form, which could become sporicidal also if the spores were heated to 50 degrees C. B. megaterium spores mixed with 0.1% (32.6 mM) H(2)O(2) solution appeared to absorb as much as 15 micromol/mg dry weight or about 0.5 mg/mg, but only a third to half of the peroxide could be recovered by water washing. A part of the unrecovered peroxide was degraded in reactions resulting in measurable production of oxygen. Degradation was not reduced by heating the spores to 65 degrees C or by azide and so appeared to be non-enzymatic. Spores of the anaerobe Clostridium sporogenes were also sensitized to ultraviolet killing by H(2)O(2) treatment followed by drying. They appear to absorb less peroxide, only about 2 micromol/mg, but had lower capacities to degrade H(2)O(2) so that nearly all of the peroxide could be recovered by washing with water. The findings presented should be helpful in the design of new methods for synergistic killing of spores by H(2)O(2) and UV irradiation or dry heat, especially involving, for example, packaging materials.     

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.     

The influence of water on the resistance of spores to inactivation by gaseous ethylene oxide

Standardized conditions for exposure to ethylene oxide were used to evaluate the resistance of spores dried for various times at different relative humidities and temperatures. Spores dried under conditions of high humidity exhibited low resistance to the sterilant, the resistance increasing as the relative humidity (RH) was decreased. Increasing the temperature of drying amplified this effect by reducing the time required for equilibration to a specific RH. Spores dried over a desiccant at 37°C showed a slight rise followed by a fall in resistance. Spores maintained under these conditions for a long period of time increased in resistance. Spores rapidly dried by exposure to low RH, over a desiccant or at elevated temperature, dried unevenly resulting in a heterogeneous population with respect to ethylene oxide resistance. This was expressed as non-logarithmic survivor curves. The initial vacuum drawn influences resistance. The resistance of spores dried on aluminium foil increased as the pressure was reduced. The rate at which the pressure was reduced had little effect on resistance, except with highly desiccated spores. Dried spores held at different reduced pressures with humidification, showed no differences in resistance. The implications of these findings in relation to the operation of ethylene oxide sterilization cycles and the preparation and use of biological monitors is discussed.