Rapid colorimetric technique for the enumeration of fecal coliforms in water

Abstract

A new colorimetric method for the detection of fecal coliforms (FC) in water has been developed. The method is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-8-D-galactoside (ONPG) by FC. This technique provides an estimate of the FC concentration within 8 to 20 hr. One hundred ml of test sample is passed through a 0.45 µm membrane filter. This filter is then incubated at 37 C for one hr in EC medium followed by the addition of filter sterilized ONPG. The incubation is continued at 44.5 C until 1/2 maximum absorbance (420 nm) is reached. The time between the start of incubation and the 1/2 maximum absorbance is proportional to the concentration of FC 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, using samples which ranged from grossly polluted streams to relatively clean lake water, a linear relation between ONPG hydrolysis times and FC MPN values was established.