Rapid detection, identification, and enumeration of Escherichia coli cellsin municipal water by chemiluminescent in situ hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simulta neous detection, identification, and enumeration of culturable Escherichia coli cells in 100 mi of municipal water within one working day. Following f iltration and 5 h of growth on tryptic soy agar at 35 degreesC, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membran e filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each mic rocolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent sub strate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selec tion based on 16S ribosomal DNA (rDNA) sequence alignments and sample matri x interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only th e rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C wit h the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strai ns and 17 other bacterial species, including closely related species. No ba cterial strains other than E. coli and Shigella spp. were detected, which i s in accordance with 16S rDNA sequence information. Furthermore, the enumer ation of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH em ploys traditional membrane filtration and culturing techniques while provid ing the added sensitivity and specificity of PNA probes in order to yield f aster and more definitive results.