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 degrees 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.     

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.     

The relationship between the age of bacillus subtilis spores and their resistance to ethylene oxide

ИААлeдуя стeрилизуящee дeйсBниe газообразного этилeноксида на споры бактeАии Bacillus subtilis, мы обнаружили возрастныe разлизия в устойчивости спор, полуGeнных из одной и тоHй жe субкультуры. Это наблядeниe доказываeт, чРо послe окончания eазвития из вeгeтативной клeтки споры продолжают подвeргаться глкбоким цитологичeским чзмeнeниям — и нeзависимо от влияния внeшнeй срeды.SS     

Germination of spores of Bacillus subtilis var. niger following exposure to gaseous ethylene oxide

Spores of Bacillus subtilis var. niger exposed to a lethal dose of ethylene oxide (ETO) germinated freely under a variety of nutritional conditions. Outgrowth, however, did not occur. Good germinants for ETO-exposed spores were (in order of decreasing effectiveness) alanine, valine, cysteine, isoleucine and histidine. Asparagine, while a germinant for unexposed spores, did not allow the germination of ETO-exposed spores. Mixtures of amino acids were no more effective than alanine alone. Exposure to ETO lowered both the rate and amount of germination but the effect was much less than that on viability. A linear relationship was obtained by plotting germination in glucose alanine against survivors on a logarithmic scale. This relationship did not occur when valine was the germinant.     

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.     

Modeling the inactivation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spores by ethylene oxide processing

Ethylene oxide is currently a dominant agent in medical device sterilization. This work intends to study the main effects and interactions of temperature, ethylene oxide concentration, and relative humidity on commercial spore strips of Bacillus subtilis, var. niger (ATCC 9372) inactivation, the most common microorganism used in controlling the efficacy of the process. Experiments were carried out using a full factorial experimental design at two levels (2³ factorial design). Limit target exposure conditions for ethylene oxide concentration, temperature, and relative humidity were 250–1,000 mg EO/l, 40–60°C, and 50–90%, respectively. Adopting a different approach from the first-order kinetics, a Gompertz model was successfully applied in data fitting of the inactivation curves. Bacillus subtilis kinetic behavior presented a sigmoidal inactivation with an initial shoulder (λ), followed by a maximum inactivation rate (k_max), these being model parameters. It was concluded that temperature and ethylene oxide concentration were the most significant factors and consequently, additional experiments were carried out aiming at describing the parameters' dependence on these process factors. Mathematical relations describing such dependences were successfully developed and included in the Gompertz kinetic model. The predictive ability of this integrated model was assessed, and its adequacy in predicting B. subtilis inactivation was proven.     

Inactivation of Bacillus spores by gaseous ozone

The sporicidal activity of ozone in the gas phase was investigated. Spores of six strains of Bacillus species deposited on filter paper or glass fibre filter were conditioned at different relative humidities (r.h.), and then exposed to ozone ranging in concentration from 0.5 to 3.0 mg/l at different r.h. There was a lag phase in the initial stage of exposure followed by an exponential decrease in the number of survivors with time, although no lag phase was observed with one strain. Inactivation rates increased with increasing exposure r.h. while no significant inactivation was attained at a r.h. of 50% or below. The conditioning r.h. influenced the duration of the lag phase. The D-values (decimal reduction time) in the logarithmic phase varied roughly in inverse proportion to the ozone concentration.     

Inactivation of Bacillus spores by gaseous ozone

The sporicidal activity of ozone in the gas phase was investigated. Spores of six strains of Bacillus species deposited on filter paper or glass fibre filter were conditioned at different relative humidities (r.h.), and then exposed to ozone ranging in concentration from 0.5 to 3.0 mg/1 at different r.h. There was a lag phase in the initial stage of exposure followed by an exponential decrease in the number of survivors with time, although no lag phase was observed with one strain. Inactivation rates increased with increasing exposure r.h. while no significant inactivation was attained at a r.h. of 50% or below. The conditioning r.h. influenced the duration of the lag phase. The D-values (decimal reduction time) in the logarithmic phase varied roughly in inverse proportion to the ozone concentration.     

Factors influencing the resistance of biological monitors to ethylene oxide

The resistance of bacterial spore monitors is markedly influenced by the environmental conditions existing during development of the spores and, subsequently, in the preparation and evaluation of the monitor. Sporulation medium, suspending medium, pasteurization and storage conditions influence resistance of spores of Bacillus subtilis var. niger to ethylene oxide, but incubation temperature and age of sporulating culture appear to be unimportant. The conditions under which the spore suspension is dried on the supporting medium of the monitor exerts a major influence on resistance. Spores exposed to ethylene oxide are abnormally susceptible to damage by shaking with Ballotini, a method frequently used to recover spores from monitors. Nutritional conditions, pH and temperature of incubation influence the ability of survivors to form colonies on solidified media.     

The metabolism of ethylene to ethylene oxide in cultivars of Vicia faba: Relationship with waterlogging resistance

The ability of cultivars of Vicia faba L. to metabolize ethylene and the resistance of such cultivars to waterlogging was assessed. Those cultivars showing the highest resistance to waterlogging also had the ability to rapidly metabolize ethylene. It is suggested that a causal relationship may exist between the two processes.     

Modeling the inactivation kinetics of bacillus spores by glutaraldehyde

Aims: Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. Methods and Results: We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality‐controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. Conclusions: The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time‐duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. Significance and Impact of the study: The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde‐based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.     

Aeration time following ethylene oxide sterilization for reusable rigid sterilization containers: concentration of gaseous ethylene oxide in containers

Because ethylene oxide (EO) gas is toxic to humans, restrictions have been imposed on its use for sterilization, specifying allowable levels of residual EO remaining in sterilized apparatus and materials. However, the aeration time that optimizes the removal of the remaining EO when a rigid sterilizing container is used for a vessel had not been identified. Therefore, polyvinyl chloride, which easily adsorbs EO, was placed in rigid sterilizing containers, and aeration was carried out after 1, 8, 12, 17, and 24 hours. After standard EO sterilization, the EO concentrations remaining in the air in the rigid containers were measured. The results indicate that a period of 17 hours of aeration is appropriate when a rigid sterilizing container is used.     

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.     

Influence of the surface temperature of packaging specimens on the inactivation of Bacillus spores by means of gaseous H(2) O(2)

Aims: To determine the influence of condensation as a function of the surface temperature of aseptic packaging, on the inactivation of Bacillus spores [Bacillus subtilis (DSM 347), B. subtilis SA22, Bacillus atrophaeus] having different surface properties by means of vaporized H₂O₂. Methods and Results: The packaging specimens inoculated with Bacillus spores were tempered and subsequently exposed to H₂O₂‐vapour. During the exposure, surface temperature curves were measured and the spore survival was determined. Results showed that decreasing the initial surface temperature of the packaging specimens had a positive effect on the sporicidal activity of H₂O₂‐vapour, where the effect was less pronounced for less hydrophilic spores. The surfaces of spores were characterized by means of the water contact angle. Conclusions: For starting surface temperatures below the dew point temperature of the sterilant gas, the condensation of highly concentrated liquid H₂O₂ on the packaging surface accelerates the killing of the spores, while the inferior wettability of more hydrophobic spores compared to more hydrophilic ones diminishes the effect. Significance and Impact of the Study: Regarding industrial packaging sterilization, a mixed microflora has to be inactivated. Promoting the condensation of H₂O₂ improves in general the killing of different species of spores, however, at various degrees depending on the wettability of spores.