A simple method for the exposure of micro‐organisms to ethylene oxide on membrane filters in a modified desiccator has been devised and used to study microbial resistance to the gaseous sterilant and the term ‘R‐value’ is suggested to express this. The resistance of many known species and isolates has been assessed and compared. Several species of Bacillus were isolated from natural habitats and their spores were found to be more resistant than the strain of Bacillus subtilis var. niger (NCTC 10073) frequently used to monitor ethylene oxide sterilization. However, endospores of some bacterial species exhibited little resistance. Fungal spores and vegetative bacteria exhibited low resistance to the sterilant except after drying in organic material when they appeared more resistant than spores of B. subtilis var. niger. It was concluded that resistance to ethylene oxide did not correlate with resistance to heat, irradiation or other chemical disinfectants, or to the existence in the endospore form per se.
The resistance of bacterial spore monitors is markedly influenced by the environmental conditions existing during development of 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.
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.
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.
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.
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