Enzymatic Detection of Coliforms and Escherichia coli within 4 Hours

Abstract

Commercial enzymatic membrane filtration (MF) tests for total coliforms (TC) and Escherichia coli in drinking water, based on ß-galactosidase (TC) or ß-glucuronidase (E. coli) detection take 18–24 h to complete. They either use chromogenic substrates (e.g., X-gal, X-gluc) or fluorogenic ones, particularly 4-methylumbelliferyl-glycosides. The latter afford improved detectability and, under particular conditions reduce the detection time to approximately 8 h. As part of our efforts to further enhance the speed of enzymatic MF tests, we examined two more advanced, two-step approaches. The first one involves microcolony formation in a short growth phase, followed by a separate enzyme assay using 4-trifluoromethylumbelliferyl-glycosides plus a membrane permeabilizer, and reading under a UV-lamp. For E. coli this type of test takes 5.5 h but field studies revealed a high false-negative rate. Switching from visual microcolony detection to instrumental single cell detection with the aid of a laser scanner (ChemScan) resulted in tests for E. coli and TC of 3.5 h. Both procedures involve enzyme induction (3 h) in the target cells, followed by cell labelling (30 min) using fluorescein-di-ß-D-glycosides and laser scanning (3 min) of the membrane filter. Application of the ChemScan E. coli test to naturally contaminated well and surface, as well as uncontaminated well and tap (n=149) water samples has indicated >90 % agreement, and equivalence with reference methods. The false-negative rate was 3.0–6.0% versus Chromocult Coliform and m FC agar, respectively. In 10 out of 149 samples, low numbers (1–3) of the target organisms were detected by laser scanning and not by conventional plate methods. These one-working day tests for TC and E. coli would be particularly useful for emergency monitoring of drinking water.