Use of alternative carrier materials in AOAC Official Method 2008.05, efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface, quantitative three-step method

Abstract

The quantitative Three-Step Method (TSM) for testing the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface (glass) was adopted as AOAC Official Method 2008.05 in May 2008. The TSM uses 5 x 5 x 1 mm coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers and neutralization, spores are removed from carriers in three fractions (gentle washing, fraction A; sonication, fraction B; and gentle agitation, fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. The counts are summed over the three fractions to provide the density (viable spores per carrier), which is log10-transformed to arrive at the log density. The log reduction is calculated by subtracting the mean log density for treated carriers from the mean log density for control carriers. This paper presents a single-laboratory investigation conducted to evaluate the applicability of using two porous carrier materials (ceramic tile and untreated pine wood) and one alternative nonporous material (stainless steel). Glass carriers were included in the study as the reference material. Inoculated carriers were evaluated against three commercially available liquid sporicides (sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde), each at two levels of presumed efficacy (medium and high) to provide data for assessing the responsiveness of the TSM. Three coupons of each material were evaluated across three replications at each level; three replications of a control were required. Even though all carriers were inoculated with approximately the same number of spores, the observed counts of recovered spores were consistently higher for the nonporous carriers. For control carriers, the mean log densities for the four materials ranged from 6.63 for wood to 7.14 for steel. The pairwise differences between mean log densities, except for glass minus steel, were statistically significant (P < 0.001). The repeatability standard deviations (Sr) for the mean control log density per test were similar for the four materials, ranging from 0.08 for wood to 0.13 for tile. Spore recovery from the carrier materials ranged from approximately 20 to 70%: 20% (pine wood), 40% (ceramic tile), 55% (glass), and 70% (steel). Although the percent spore recovery from pine wood was significantly lower than that from other materials, the performance data indicate that the TSM provides a repeatable and responsive test for determining the efficacy of liquid sporicides on both porous and nonporous materials.