Multiregional Evaluation of the SimPlate Heterotrophic Plate Count Method Compared to the Standard Plate Count Agar Pour Plate Method in Water

Abstract

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35°C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r ‫؍‬ 0.95; y ‫؍‬ 0.99X ؉ 0.06).Water utilities are required to maintain a detectable disinfection residual in water distribution systems or measure for heterotrophic plate count (HPC) bacteria (6). The standard HPC pour plate method is an approved U.S. Environmental Protection Agency USEPA method (5) for reporting HPC in lieu of testing for residual disinfectant concentration or for testing when residual disinfectant levels are less than 0.2 mg/ liter in finished waters (4). This method, as well as other HPC methods, such as membrane filtration or spread plating, may be also used to collect data for internal purposes (nonreporting). All the methods (1) for testing of heterotrophic bacteria require time-consuming preparation of media and can be difficult to read. Recently, the SimPlate total plate count method for determining the most probable number (MPN) of microorganisms in food was developed by IDEXX Laboratories, Westbrook, Maine (3), and approved by the Association of Official Analytical Chemists (AOAC) International Research Institute (2). The formulation was modified to allow for the detection of heterotrophic bacteria in water. The test known as SimPlate for HPC medium contains substrates that are hydrolyzed by microbial enzymes to release 4-methylumbelliferone, which fluoresces blue under a long-wavelength (365-nm) 6-W UV light after incubation for 48 h at 35°C. The bacteria are detected as fluorescent wells on the SimPlate. The bacterial density of a water sample is determined by determining the number of positive wells and by using the MPN table provided for SimPlate. This format will allow a MPN/milliliter value up to 738 without any dilution. The objective of this study was to compare the performance of SimPlate and the HPC pour plate method (1) for the enumeration of heterotrophic bacteria. Six laboratories in different regions of the United States participated in this study.Between May and June 1997, naturally occurring heterotrophic bacterium samples were collected in sterile vessels at each site. The samples consisted of chlorinated drinking waters (neutralized with sodium thiosulfate) (1), well waters, untreated natural (raw) waters (lakes and streams), and secondary chlorinated sewage effluents (neutralized with sodium thiosulfate). Since chlorinated drinking waters have relatively few or no heterotrophic bacteria, the sites prepared composites of raw waters and/or secondary effluent with neutralized chlorinated drinking water. The composite samples were prepared in ratios of 1:1, 1:2, and 2:1 to allow as broad a range of bacterial counts as possible to be represented in the study. Each site was requested to test 100 samples for the evaluation. Approximately 40% of the samples were to be from ...

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