Determining the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard nonporous surface using the quantitative three step method: collaborative study

J AOAC Int. Jul-Aug 2008;91(4):833-52.


A collaborative study was conducted to validate the quantitative Three Step Method (TSM), a method designed to measure the performance of liquid sporicides on a hard nonporous surface. Ten laboratories agreed to participate in the collaborative study; data from 8 of 10 participating laboratories were used in the final statistical analysis. The TSM uses 5 x 5 x 1 mm glass coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure to a test chemical and a neutralization agent, spores are removed from carriers in 3 fractions: passive removal (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. Control counts are compared to the treated counts, and the level of efficacy is determined by calculating the log10 reduction (LR) of spores. The main statistical goals were to evaluate the repeatability and reproducibility of the LR values, to estimate the components of variance for LR, and to assess method responsiveness. AOAC Method 966.04-Method II was used as a reference method. The scope of the validation was limited to testing liquid formulations against spores of Bacillus subtilis, a surrogate for virulent strains of B. anthracis, on a hard nonporous surface (glass). The test chemicals used in the study were sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde. Each test chemical was evaluated at 3 levels of presumed efficacy: high, medium, and low. Three replications were required. The TSM was validated as it successfully met the statistical parameters for quantitative test methods. Satisfactory validation parameters, such as the repeatability standard deviation (Sr) and reproducibility standard deviation (SR), were obtained for control carrier counts and LR values. Both the TSM and the reference method were responsive to the efficacy levels of the test chemicals. For the 72 total TSM tests conducted, the mean (+/- standard error of the mean) log density of spores per control carrier was 6.86 (+/- 0.08); the Sr and SR were low at 0.15 and 0.27, respectively. Across the range of test chemicals, the Sr and SR estimates associated with LR were also acceptably low. The Sr ranged from 0.17 to 0.72 and the SR ranged from 0.34 to 1.43. Overall, the Sr and SR estimates associated with the efficacy data were within the ranges published for other quantitative methods and meet the performance characteristics necessary for validation.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Analysis of Variance
  • Anti-Bacterial Agents / pharmacology*
  • Bacillus anthracis / drug effects
  • Bacillus subtilis / drug effects*
  • Biological Assay
  • Calibration
  • Culture Media
  • Disinfectants / pharmacology
  • Glutaral / pharmacology
  • Hydrochloric Acid / pharmacology
  • Indicators and Reagents
  • Oxidants / pharmacology
  • Peracetic Acid / pharmacology
  • Quality Control
  • Reference Standards
  • Reproducibility of Results
  • Sodium Hypochlorite / pharmacology
  • Spores, Bacterial / drug effects*
  • Surface Properties
  • Water Microbiology


  • Anti-Bacterial Agents
  • Culture Media
  • Disinfectants
  • Indicators and Reagents
  • Oxidants
  • Sodium Hypochlorite
  • Peracetic Acid
  • Hydrochloric Acid
  • Glutaral