Development of reproducible test inocula for disinfectant testing

For the development and approval of disinfectants, laboratory tests which combine repeatability and reproducibility with relevance to practical conditions are required to ensure optimum standards of efficacy under use conditions. Over the years, although an increasingly rigorous approach has been adopted in devising European and US Standard Test Methods for disinfectants, which includes specifying all aspects of test methodology, the precision of these methods remains a matter for concern. Studies of proposed European test methods indicate that, although some of the variability is methodological in origin, or is derived from errors in preparing test solutions and performing the tests, one of the major sources of error is lack of reproducibility in the performance of the test inoculum. Results indicate that day to day variability in biocide sensitivity of sequential subcultures arises not only from variations in the phenotype generated from the laboratory stock culture, but also from lack of standardization of conditions used to harvest and prepare test inocula. Further reductions in reproducibility between test periods both within or between laboratories probably arise from alterations in the genotype of the laboratory stock culture or source culture during storage under conditions of refrigeration, freezing or freeze-drying. Methods for production of reproducible inocula are considered and some studies aimed at development of test inocula with more reproducible biocide resistance are described.