Neutron and y-Ray Radiation Killing of Bacillus Species Spores: Dosimetry, Quantitation, and Validation Techniques

The validation of sterilization processes is currently monitored by qualitative estimates of bacterial spores using inactivation of commercially available spore-impregnated cellulose fiber strips where sterility is determined by the presence or absence of growth in an appropriate medium. Using the standardized technique in the U.S. Pharmacopeia (USP) National Formulary XXII, spore kill is quantified by pulping multiple spore strips in a large volume of diluent. Such tests do not allow quantitative analysis of killing on each separate strip. In this study, the standard USP test was modified to quantify sterilization of spores on single cellulose fiber strips that were irradiated with fission neutrons produced by a TRIGA reactor and by 60Co y-rays. The technique, developed at the Armed Forces Radiobiology Research Institute (AFRRI), was validated for similarly treated spore strips by comparing data obtained from an independent commercial laboratory and certificated values provided by the supplier of the spore strips. The number of Bacillus subtilis and Bacillus pumilus spores identified by the two laboratories were similar for controls (unirradiated spore strips) and for spore strips that received selected doses of fission neutrons or y-rays. It was thus concluded that assay techniques, developed and used at AFFRI to enumerate spore survival, accurately quantify spore kill after neutron and y-irradiation. These techniques can be used in more extensive studies to determine the killing effect for spores irradiated under a variety of environmental conditions.