Rapid detection, identification, and enumeration of Escherichia coli by fluorescence in situ hybridization using an array scanner

A new fluorescence in situ hybridization (FISH) method using peptide nuclei c acid (PNA) probes and an array scanner for rapid detection, identificatio n, and enumeration of Escherichia coli is described. The test utilizes Cy3- labeled peptide nucleic acid (PNA) probes complementary to a specific 16S r RNA sequence of E. coli. Samples were filtered and incubated fur 5 h, the m embrane filters were then analyzed by fluorescence in situ hybridization an d results were visualized with an array scanner. Results were provided as f luorescent spots representing. coli microcolonies on the membrane filter su rface. The number of fluorescent spots correlated to standard colony counts up to 100 colony-forming units per membrane filter. Above this level, bett er accuracy was obtained with PNA FISH due to the ability of the scanner to resolve neighboring microcolonies, which were not distinguishable as indiv idual colonies once they were visible by eye. (C) 2001 Elsevier Science B.V , All rights reserved.