Responding to a need for a guide for conducting Official Method validation studies of microbiological methods, AOAC utilized the experience of three microbiologists who have been active in the field of method validation. In collaboration, a document was prepared which covered the following areas: terms and their definitions associated with the Official Methods program (e.g., reference methods, alternative methods, and ruggedness testing), protocols and validation requirements for qualitative methods versus those for quantitative methods, the concept of the precollaborative study, ruggedness testing, tests for significant differences, performance indicators, and the approval process. After its preparation, this document was reviewed by the members of the Methods Committee on Microbiology and Extraneous Materials and by members of the Official Methods Board. Herein is presented the approved version of that document.

The relative efficacy of the SimPlate® Total Plate Count–Color Indicator (TPC–CI) method (SimPlate 35°C) was compared with the AOAC Official Method 966.23 (AOAC 35°C) for enumeration of total aerobic microorganisms in foods. The SimPlate TPC–CI method, incubated at 30°C (SimPlate 30°C), was also compared with the International Organization for Standardization (ISO) 4833 method (ISO 30°C). Six food types were analyzed: ground black pepper, flour, nut meats, frozen hamburger patties, frozen fruits, and fresh vegetables. All foods tested were naturally contaminated. Nineteen laboratories throughout North America and Europe participated in the study. Three method comparisons were conducted. In general, there was <0.3 mean log count difference in recovery among the SimPlate methods and their corresponding reference methods. Mean log counts between the 2 reference methods were also very similar. Repeatability (sr) and reproducibility (sR) standard deviations were similar among the 3 method comparisons. The SimPlate method (35°C) and the AOAC method were comparable for enumerating total aerobic microorganisms in foods. Similarly, the SimPlate method (30°C) was comparable to the ISO method when samples were prepared and incubated according to the ISO method.

The relative effectiveness of the SimPlate Yeast and Mold-Color Indicator method (Y&M–CI) was compared to the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) method and the proposed International Organization for Standardization (ISO) method, ISO/CD 21527, for enumerating yeasts and molds in foods. Test portions were prepared and incubated according to the conditions stated in both the BAM and ISO methods. Six food types were analyzed: frozen corn dogs, nut meats, frozen fruits, cake mix, cereal, and fresh cheese. Nut meats, frozen fruits, and fresh cheese were naturally contaminated. All other foods were artificially contaminated with either a yeast or mold. Seventeen laboratories throughout North America and Europe participated in the study. Three method comparisons were conducted. In general, there was <0.3 mean log count difference in recovery between the SimPlate method and the 2 corresponding reference methods. Moreover, mean log counts between the 2 reference methods were also very similar. The repeatability (sr) and reproducibility (sR) standard deviations were comparable between the 3 method comparisons. These results indicate that the BAM method and the SimPlate method are equivalent for enumerating yeast and mold populations in foods. Similarly, the SimPlate method is comparable to the proposed ISO method when test portions are prepared and incubated as defined in the proposed ISO method.

The relative effectiveness of the SimPlate® Coliform and E. coli Color Indicator (CEc-CI) method was compared to the AOAC 3-tube Most Probable Number (MPN) methods for enumerating and confirming coliforms and Escherichia coli in foods (966.23 and 966.24). In this study, test portions were prepared and analyzed according to the conditions stated in both the AOAC methods and SimPlate directions for use. Six food types were artificially contaminated with coliform bacteria and E. coli: frozen burritos, frozen broccoli, fluid pasteurized milk, whole almond nut meats, cheese, and powdered cake mix. Method comparisons were conducted. Overall, the SimPlate method demonstrated <0.3 log difference for total coliform and E. coli counts compared to the AOAC reference methods for the majority of food types and levels analyzed. In all cases, the repeatability and reproducibility of the SimPlate CEc-CI method were not different from those of the reference methods and in certain cases, were statistically better than those of the AOAC 3-tube MPN methods. These results indicate that the SimPlate CEc-CI method and the reference culture methods are comparable for enumeration of both total coliforms and E. coli in foods.

A new enzyme immunoassay (EIA) method for detection of motile and non-motile Salmonella was examined in a comparative study. This method uses a proprietary formulation of polyclonal antibodies to Salmonella and is controlled to maintain specificity. Sensitivity is enhanced with an additional antibody reaction designed to minimize false-negative reactions attributable to steric interference that can occur during conjugate binding in immunoassay procedures. Twenty food types representative of a wide variety of food products were analyzed by both the EIA method and the AOAC/Bacteriological Analytical Manual (BAM) method, 967.26. Of the 1000 samples analyzed, there was a 95.6% agreement rate between the EIA method and the AOAC/BAM method. False-negative rates for the 2 methods were comparable for all foods and all Salmonella levels except ground poultry, where the EIA method detected significantly more confirmed positive samples than did the AOAC/BAM method. Twenty-seven samples were positive by EIA but negative by the culture method, and 17 samples were negative by EIA but positive by the culture method. There were no false-positive isolates detected in the comparative study.