Sporicidal testing of commercial germicides using a chemical standard and a calibrated bioindicator

The AOAC sporicidal method uses as a standard the resistance of spores on carriers to 2.5N HCl. This resistance is variable at exposure times ranging from 2 to 20 min. The method described in this paper uses a glutaraldehyde standard and distinguishes various levels of sporicidal activity in the presence of 1-5% glutaraldehyde by using appropriate spore strains, spore preparations, and spore levels. The resistances of 2 Bacillus subtilis 19659 spore preparations cultured in 10% Columbia broth plus manganese and nutrient agar plus minerals, as well as that of B. subtilis var. niger cultured on Lab-Lemco agar, were tested. T-soy broth was a better recovery medium than fluid thioglycollate or modified fluid thioglycollate for B. subtilis 19659 spores exposed to HCl. Sporicidal tests were done on B. subtilis 19659 spores with 2 types of spore preparations. A commercial glutaraldehyde germicide was used for comparison of the sporicidal activity of the glutaraldehyde standard. Two strains of B. subtilis spores and 4 levels of spores (20,000-80,000, 100,000-400,000, 500,000-800,000, and 1,000,000 and up) were removed from check penicylinders from the same batches used for sporicidal tests. B. subtilis var. niger spores were the most resistant to HCl, while B. subtilis 19659 spores were more resistant to glutaraldehyde. Sporicidal activities of a commercial germicide containing 2.5% glutaraldehyde with additives and another containing 5% glutaraldehyde in phosphate buffer were similar. Both totally destroyed high levels of B. subtilis 19659 spores cultured in 10% Columbia broth plus manganese. Results indicate that use of a glutaraldehyde standard, calibrated numbers of spores on penicylinders (bioindicators), and appropriate spore strains and preparations can reduce the variability of sporicidal testing of commercial germicides.