Enliven: Microbes and Microbial Techniques

The discovery of chromogenic and fluorogenic substances for their application in tests has brought about huge advantages over the use of tests based on traditional culture media. In the field of drinking water microbiology, many studies are addressed at reviewing methods of analysis of coliform bacteria and Escherichia coli. The main objective of this study was to evaluate a new method of detection and quantification of total coliforms and E. coli in water by the Most Probable Number (MPN) technique using as broth culture a chromogenic-fluorogenic medium (CCL) according to the fundamental aspects of the validation guidelines of qualitative and quantitative methods of AOAC International and the standard ISO 16140 by analyzing 99 water amples from different origins. The new method is based on the splitting of specific substrates for glucuronidase and tryptophanase activity by E. coli, and for β-galacosidase by E. coli and coliform bacteria, and also on the gas production by the coliform group. A method described by ISO 9308-3 was choosed as reference, which is based on the fermentation of lactose with acid production, on the oxidase-negative character of total coliforms (for confirmation) and, additionally, on the production of indole from tryptophan at 44°C (E. coli confirmation). By different validation methodologies of alternative methods it was proven that the new chromogenic fluorogenic method using the Most Probable Number, employing methylumbelliferyl-β-D-glucuronide and ortho-nitrophenyl-β-D-galactopyranoside as enzymatic markers for the detection and counting of coliforms and E. coli, is equivalent to the procedures described in the ISO reference method for the same purpose in terms of diagnostic accuracy and precision of the count. The composition of the culture medium used allowed for the correct determination of the target microorganisms. The new procedure allowed obtaining results in less time, with less reagents, and more easily.