Rapid Detection of Starved Escherichia coli with Respiratory Activity in Potable Water by Signal-Amplified in situ Hybridization Following Formazan Reduction.

The aim of this study was to develop a rapid method for the specific detection of respiring Escherichia coli (an indicator of fecal contamination) in potable water. Fluorescence in situ hybridization (FISH) with a rRNA-targeted oligonucleotide probe was used to detect E. coli cells and bacterial respiratory activity was estimated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). Fluorescent signals from hybridized cells were increased by optimized tyramide signal amplification (TSA). Respiring E. coli in potable ground water with low rRNA content were enumerated within 8 hours using signal-amplified in situ hybridization following formazan reduction (TSA-CTC-FISH), whereas these starved E. coli cells could not be detected by conventional FISH (FISH without signal amplification) which generated weak fluorescence. TSA-CTC-FISH can be used for simultaneous identification in situ based on phylogenetic information and the activity of individual bacterial cells in potable water. This method would be useful in the rapid monitoring of harmful or fecal indicator bacteria in potable water.