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A specially built thermochemical death-rate apparatus is described which can be used to determine the resistance of microorganisms to ethylene oxide under controlled conditions. The apparatus was designed to provide instantaneous exposure of microorganisms to ethylene oxide and to eliminate variables that could result in errors when death kinetic reaction rates are calculated. The apparatus is used to obtain ethylene oxide resistance data which are useful in evaluating and developing sterilizing cycles for materials with known bacterial concentrations, as well as for calculating probability factors on which a given test condition can be expected to provide sterilization.

Spores of Bacillus subtilis var. niger, dried on nonhygroscopic and hygroscopic surfaces, were enclosed in one of four thicknesses of low-density polyethylene film (2, 4, 6, and 20 mils). The surfaces were then placed in a specially designed thermochemical death rate apparatus and exposed to an ethylene oxide concentration of 600 mg/liter (at 54.4 C) and 50% relative humidity. Survival data, including both spore survivor curves and decimal reduction values (expressed as D values at 54.4 C-600 mg of ethylene oxide per liter), demonstrated similar survivor patterns when the B. subtilis var. niger spores were enclosed in low-density polyethylene films 2, 4, and 6 mils thick. A comparison of these patterns with those of spores enclosed in glassine and subjected to identical exposure conditions revealed only slight variations. The use of 20-mil polyethylene film greatly increased the time required to kill B. subtilis var. niger spores under the exposure conditions.

The death rate kinetics of several sporeforming and nonsporeforming microorganisms, including radiation-resistant cocci, were determined by exposing them to a mixture of ethylene oxide and dichlorodifluoromethane (500 mg of ethylene oxide per liter, 30 to 50% relative humidity, and 54.4 C). Spore survivor curves obtained from tests of inoculated and exposed hygroscopic and nonhygroscopic carriers showed that the spores of Bacillus subtilis var. niger are more resistant to ethylene oxide than are spores of Clostridium sporogenes, B. stearothermophilus, and B. pumilus. The decimal reduction times (expressed as D values at 54.4 C-500 mg of ethylene oxide per liter) obtained under the test conditions for B. subtilis var. niger spores on hygroscopic and nonhygroscopic carriers exceeded the values obtained for the other organisms considered, both sporeformers and nonsporeformers. The decimal reduction times for the vegetative cells of the radiation-resistant organisms (Micrococcus radiodurans and two strains of Streptococcus faecalis) and the ATCC strain of S. faecalis demonstrated comparable resistance to ethylene oxide with the spores of C. sporogenes, B. stearothermophilus, and B. pumilus, but not those of B. subtilis var. niger.

An investigation determined the effects of environmental moisture content or water activity (Aw), exposure humidity, and sterilant concentration on the resistance of microbial spores. Decimal reduction values [expressed as D values at 54.4 C-specified concentration (milligrams per liter) of ethylene oxide] were determined from spore destruction curves of Bacillus subtilis var. niger dried on hygroscopic and nonhygroscopic surfaces. Four groups of spore preparations were preconditioned in one of four Aw environments (<0.1, 0.1, 0.5, 0.95) for 2 weeks or longer and were exposed to 500 mg of ethylene oxide per liter at 54.4 +/- 3 C and 10, 50, and 95% relative humidity in a specially designed thermochemical death rate apparatus. A fifth group did not receive any preconditioning treatment and was exposed immediately after preparation, in the same apparatus at the same temperature, to ethylene oxide concentrations of 200, 400, 600, 800, and 1,200 mg/liter and relative humidities of 15, 30, 50, 60, and 90%. The resistance of the spores on both types of surfaces to ethylene oxide increased proportionately with the Aw of the conditioning environment. The study also showed that moisture in the exposure system was not as critical a variable as the ethylene oxide concentration. The spore destruction rates, irrespective of the carrier types at all concentrations and at different humidities, varied little from one another. The decimal reduction values were reduced as the ethylene oxide concentration increased, and no optimal exposure humidity concentration was observed.