Journal of Microbiological Methods 2000-11-01

Rapid detection of fluorescent and chemiluminescent total coliforms and Escherichia coli on membrane filters.

S O Van Poucke, H J Nelis

Index: J. Microbiol. Methods 42(3) , 233-44, (2000)

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Abstract

The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-beta-D-glycosides as enzyme substrates and a UV-lamp for visualization. The most rapid procedures based on this approach for the demonstration of these indicator bacteria in water take 6-7.5 h to complete. As part of efforts to further reduce the detection time, an improved two-step procedure for the fluorescence or chemiluminescence labelling of microcolonies of E. coli/TC on a membrane filter has been developed. Essential features of this approach include a separation of the bacterial propagation and target enzyme induction from the actual enzymatic labelling, the use of improved fluorogenic, i.e., 4-trifluoromethylumbelliferyl-beta-D-glycosides and fluorescein-di-beta-D-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for beta-glucuronidase/beta-galactosidase, of enzyme inducers, of special membrane filters and of polymyxin B to promote the cellular uptake of the substrate. This labelling procedure has been applied in conjunction with different detection devices including a UV-lamp, CCD-cameras, X-ray film and the ChemScan((R)) RDI. Using the former three, microcolonies of pure cultures could be detected within 5.5-6.5 h, but waterborne E. coli/TC may fail to form microcolonies in this short time period, thus yielding poor sensitivity and a high false-negative rate. In contrast, a quantitative enumeration was feasible in less than 4 h with the ChemScan((R)) RDI, owing to its ability to detect both microcolonies and non-dividing single cells.