Tomasino Stephen F, Parker Albert E, Hamilton Martin A, Hamilton Gordon C
U.S. Environmental Protection Agency, Office of Pesticide Programs, Biological and Economic Analysis Division, Microbiology Laboratory Branch, Environmental Science Center, Fort Meade, MD 20755-5350, USA.
J AOAC Int. 2012 Nov-Dec;95(6):1618-28. doi: 10.5740/jaoacint.12-170.
The U.S. Environmental Protection Agency (EPA), in collaboration with an industry work group, spearheaded a collaborative study designed to further enhance the AOAC use-dilution method (UDM). Based on feedback from laboratories that routinely conduct the UDM, improvements to the test culture preparation steps were prioritized. A set of modifications, largely based on culturing the test microbes on agar as specified in the AOAC hard surface carrier test method, were evaluated in a five-laboratory trial. The modifications targeted the preparation of the Pseudomonas aeruginosa test culture due to the difficulty in separating the pellicle from the broth in the current UDM. The proposed modifications (i.e., the modified UDM) were compared to the current UDM methodology for P. aeruginosa and Staphylococcus aureus. Salmonella choleraesuis was not included in the study. The goal was to determine if the modifications reduced method variability. Three efficacy response variables were statistically analyzed: the number of positive carriers, the log reduction, and the pass/fail outcome. The scope of the collaborative study was limited to testing one liquid disinfectant (an EPA-registered quaternary ammonium product) at two levels of presumed product efficacies, high and low. Test conditions included use of 400 ppm hard water as the product diluent and a 5% organic soil load (horse serum) added to the inoculum. Unfortunately, the study failed to support the adoption of the major modification (use of an agar-based approach to grow the test cultures) based on an analysis of method's variability. The repeatability and reproducibility standard deviations for the modified method were equal to or greater than those for the current method across the various test variables. However, the authors propose retaining the frozen stock preparation step of the modified method, and based on the statistical equivalency of the control log densities, support its adoption as a procedural change to the current UDM. The current UDM displayed acceptable responsiveness to changes in product efficacy; acceptable repeatability across multiple tests in each laboratory for the control counts and log reductions; and acceptable reproducibility across multiple laboratories for the control log density values and log reductions. Although the data do not support the adoption of all modifications, the UDM collaborative study data are valuable for assessing sources of method variability and a reassessment of the performance standard for the UDM.
美国环境保护局（EPA）与一个行业工作组合作，牵头开展了一项合作研究，旨在进一步改进美国官方分析化学师协会（AOAC）的使用稀释法（UDM）。根据常规进行UDM的实验室的反馈，优先对测试培养物制备步骤进行改进。在一项有五个实验室参与的试验中，评估了一组主要基于AOAC硬表面载体测试方法中规定的在琼脂上培养测试微生物的修改措施。由于在当前的UDM中难以将菌膜与肉汤分离，这些修改措施针对铜绿假单胞菌测试培养物的制备。将提议的修改措施（即改进后的UDM）与当前用于铜绿假单胞菌和金黄色葡萄球菌的UDM方法进行了比较。猪霍乱沙门氏菌未纳入该研究。目标是确定这些修改是否降低了方法的变异性。对三个效力响应变量进行了统计分析：阳性载体数量、对数减少量和通过/失败结果。合作研究的范围仅限于在假定的高、低两种产品效力水平下测试一种液体消毒剂（一种EPA注册的季铵产品）。测试条件包括使用400 ppm硬水作为产品稀释剂，并在接种物中添加5%的有机污垢负载（马血清）。不幸的是，基于对方法变异性的分析，该研究未能支持采用主要的修改措施（使用基于琼脂的方法来培养测试培养物）。在各种测试变量中，改进后方法的重复性和再现性标准偏差等于或大于当前方法。然而，作者建议保留改进后方法的冷冻储备制备步骤，并基于对照对数密度的统计等效性，支持将其作为对当前UDM的程序变更予以采用。当前的UDM对产品效力的变化显示出可接受的响应性；在每个实验室的多次测试中，对照计数和对数减少量具有可接受的重复性；在多个实验室中，对照对数密度值和对数减少量具有可接受的再现性。尽管数据不支持采用所有修改措施，但UDM合作研究数据对于评估方法变异性的来源以及重新评估UDM的性能标准具有重要价值。
