Direct Viable Count Combined with Fluorescence in situ Hybridization (DVC-FISH) for Specific Enumeration of Viable Escherichia coli in Cow Manure

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A direct viable count procedure combined with fluorescence in situ hybridization (DVC-FISH) was developed for the specific detection and enumeration of viable Escherichia coli in cow manure. The DVC method was performed by trapping bacterial cells, extracted from cow manure samples, onto Nucleopore filters followed by incubation on a DVC medium containing yeast extract and four gyrase inhibitors. E. coli cells were identified by using the probe ES445. The DVC method efficiently promoted the elongation of E. coli cells and allowed for the recognition of individual cell division events, by observing microcolonies. Cell recovery by DVC-FISH together with bacterial extraction, was 53% with an inoculum of 10(7) to 10(10) cells g(-1) dry weight, when the manure samples were inoculated with a fresh culture of E. coli and determinations were made immediately. An examination of the survival of E. coli in a cow manure microcosm showed that an increasing fraction of E. coli became non-culturable but were still detectable by DVC-FISH. All these results suggest that DVC-FISH is useful for enumerating viable, even non-culturable, E. coli in cow manure.