Rapid enumeration of fecal coliforms in water by a colorimetric β-galactosidase assay

Abstract

The colorimetric β-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-β-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 hr. A 100-ml portion of test sample was passed through a 0.45-μm membrane filter. This filter was then incubated at 37°C for 1 hr in EC medium followed by the addition of filter- sterilized ONPG. The incubation was continued at 44.5°C until a half- maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 hr. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69%), Enterobacter cloacae (2.32%), Klebsiella pneumoniae (0.66%), and Citrobacter freundii (0.33%).