Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and ␤-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and-positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670-and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the ␣-decarboxylation of L-glutamic acid to yield ␥-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-␤-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65؇C but not at 68؇C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.

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The limits of detection of 10 genotoxins representing 7 chemical classes with varying structures and modes of action were compared using the Ames test (Salmonella plate-incorporation test) with 2 tester strains, 2 standard colorimetric methods (the umu test and SOS Chromotest), and modifications of the umu and SOS Chromotests developed during the course of this study. The purpose of the study was to determine the sensitivity and reproducibility of each of the six methods. The sensitivities of the methods were compared using two criteria: the concentrations required for doubling responses, and the minimum concentrations required to produce statistically significant increases from background controls. The Ames test with strains TA98 and TA100 was ranked as the most sensitive method more often than the others, but the results indicated that the umu tests were statistically equivalent to the Ames test. The original SOS Chromotest kit method was highly sensitive in detecting the direct acting genotoxins, but neither SOS test was as sensitive as the other methods in detecting indirect acting genotoxins. The umu microtiter plate test is the least expensive of the assays and would be the most suitable for screening large numbers of environmental samples.

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Abstract--Survival of calf rotavirus and reovirus under controlled laboratory conditions in microorganism-free, distilled and wastewater at 8 and 26°C was examined by periodic measurement of cytopathic effects (CPE) and indirect fluorescent antibody (IFA) assays. Five samples of both water-types were collected and inoculated with the two viruses. Three samples of each type of water were divided into two bottles, one per virus, for incubation at 8°C. Two samples were used at 26°C, one per trial. In the absence of light and shaking at 26°C, 7 13 days were required for a loss of 90~o infectivity for rotavirus and reovirus, while at 8°C, averages were 80 days for rotavirus and 260 days for reovirus. Virus infectivity remained for more than 30 days at 26°C and 400 days at 8°C. Rates of decline were 10-100 times greater at 26 than at 8°C, but at both temperatures, the MPN log10 rate of decline of infectivity was linear.