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

Abstract

The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-β- 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-β- d-glycosides and fluorescein-di-β- d-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for β-glucuronidase/β-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 ® 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 ® RDI, owing to its ability to detect both microcolonies and non-dividing single cells.