Detection and Quantification of E. coli and Coliform Bacteria in Water Samples with a New Method Based on Fluorescence In Situ Hybridisation

Abstract

A fast and sensitive method for the specific detection of E. coli and coliform bacteria in water samples based on the Fluorescence in situ Hybridization (FISH) technology was developed by transferring the standard slide-based FISH protocol to a filter membrane-based method. For detection and quantification two different strategies were implemented and tested: (1) the direct detection of single E. coli and coliform bacterial cells on the filter membranes (single cell protocol) and (2) the detection of micro-colonies after incubation of the filter membranes on a nutrient agar plate (micro-colony protocol). Both protocols were validated using drinking water samples spiked with pure cultures as well as naturally contaminated water samples including heat treated and disinfected water samples (chlorine, UV). The single cell approach was shown to be very fast. Yet especially for low cell numbers the microscopic evaluation can become very labour-intensive. For routine application, an automated cell detection and quantification would be necessary which so far could not be implemented. Moreover, the approach showed some limitations with disinfected samples Due to the incubation step the micro-colony approach is more time consuming but it yielded very good results for all different samples and conditions tested. This approach has the potential of practical use as only a low magnification factor is needed for microscopic evaluation, making even manual quantification feasible. In addition automated quantification can be implemented. Consequently this approach can be recommended as an alternative method to detect E. coli and coliform bacteria in water samples.