Evaluation of novel enzyme substrates for the detection of coliforms in water samples

Chilvers, Kay Frances (2002) Evaluation of novel enzyme substrates for the detection of coliforms in water samples. Doctoral thesis, Northumbria University.

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Abstract

Possession of specific enzymes can be diagnostic and more reliable than detection of the end products of a metabolic pathway for the enumeration of coliform organisms. A number of chromogenic 13-galactosidase substrates were evaluated with a range of coliform organisms with regard to the intensity of the colour formed upon hydrolysis of the substrate and potential inhibitory effect on coliform growth. A dual substrate membrane filtration medium was developed to detect coliforms and E. coli on the same membrane filter, constituting a highly cost-effective method without the need for extensive confirmatory tests. The medium was evaluated with a number of coliform organisms and simulated chlorine-stressed contaminated water samples. The intense fluorescence of coumarin-based molecules has enabled them to be incorporated into enzyme-based tests for the quantitative assay of indicator bacteria. Several novel derivatives were evaluated in this study and were found to be more promising than the commercially available cournarin giving a combination of greater fluorescence over a broad pH range and reduced growth inhibition with representative coliform strains On conversion to 13-galactoside derivatives, the substrates were evaluated and incorporated into a miniaturised broth assay format, based on most probable number (MPN). The chromogenic and fluorogenic substrates described here have been evaluated and have shown their potential as powerful tools in diagnostic microbiology, utilising specific enzymatic activities of conforms, either in addition to or in place of standard methodologies.

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