Date of Award
Doctor of Philosophy
Rapid detection assays were developed for Escherichia coli by using the fluorogenic substrate 4-methylumbelliferone glucuronide (MUG). Among the Enterobacteriaceae, 97% of E. coli strains, 17% of salmonellae, and 40% of shigellae produced a (beta)-glucuronidase that cleaved MUG to produce a fluorogenic end product visible under long-wave UV light. For immediate confirmation of the presence of E. coli in Most Probable Number (MPN) tests, MUG was incorporated into Lauryl Tryptose Broth (LTB) tubes at a final concentration of 100 (mu)g/ml. Results of both the presumptive test (gas production) and confirmed test (fluorescence) were obtained from a variety of food and water samples after incubation for 24 h at 37 C. The EC test (45.5 C) for fecal coliforms confirmed over 90% of the tubes showing both gas and fluorescence. Of 17 fluorescent-positive but gas-negative tubes that were encountered, four were fluorescent because of the presence of Salmonella sp., Shigella sp. or anaerogenic strains of E. coli; the other 13 tubes must have contained (beta)-glucuronidase producers at one time, but none could be isolated after 24 h of incubation. By using the LTB-MUG MPN assay, higher counts of heat- and chlorine-injured cells were recovered, compared to the Violet Red Bile-2 agar overlay method. In addition, E. coli cells subjected to interference by antagonistic bacteria (absence of gas production) were also detected by the appearance of fluorescence. MUG was equally effective in detecting E. coli in other coliform methodologies, such as membrane filtration and direct plating, when incorporated in the culture media. A rapid confirmatory test that is amenable to automation was also developed: wells in microtitration plates were filled with a nonselective medium containing 100 (mu)g/ml of MUG. Pure or mixed cultures containing E. coli produced fluorescence after incubation for 4 h (most strains) to 24 h (a few weakly positive strains). The fluorogenic assays were very sensitive and easy to use, and the results were available much faster than results of conventional E. coli detection procedures.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Feng, Peter, "Fluorogenic assays for the detection of Escherichia coli " (1981). Retrospective Theses and Dissertations. 7419.