Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.
The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees 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 h. 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 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).
D001431:Bacteriological Techniques; D003124:Colorimetry; D004926:Escherichia coli / Q000201:enzymology / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D005696:Galactosidases / Q000378:metabolism*; D006868:Hydrolysis; D009598:Nitrophenylgalactosides / Q000378:metabolism; D014871:Water Microbiology; D014876:Water Pollution / Q000032:analysis*; D001616:beta-Galactosidase / Q000378:metabolism*
D009598:Nitrophenylgalactosides; D005696:Galactosidases; D001616:beta-Galactosidase