Direct enumeration of Escherichia coli and enteric bacteria in water, beverages and sprouts by 16S rRNA in situ hybridization

A fluorescent oligonucleotide probe complementary to a published 16S riboso mal RNA sequence of Escherichia coil was used for in situ hybridization on membrane filters for direct enumeration of cells. The direct epifluorescent filter technique (DEFT) was modified by performing a 2-h oligonucleotide h ybridization on intact cells collected on a membrane filter surface ('oligo -DEFT'). instead of the traditional acridine orange staining. The filter wa s examined by epifluorescence microscopy, and digital image analysis was us ed for the cell enumeration. Oligo-DEFT counts were linear over the range o f 10(3)-10(6) cells ml(-1) and correlated with DEFT counts (r = 0.99) The o ligo-DEFT detection limit was approximately 1 cell ml(-1). Oligo-DEFT count s correlated with standard coliform counts by membrane filtration or platin g on selective media for analysis of wafer and beverages artificially inocu lated with E, coil. Heat inactivation of the cells did not destroy their re activity in the oligo-DEFT Natural coliform populations in aquarium water w ere countable in the oligo-DEFT after a 90-min incubation of the filter on nutrient agar before the hybridization step, which allowed the dormant cell s to increase their rRNA content. Natural populations in bean sprouts were countable directly without having to perform the incubation before hybridiz ation. (C) 2000 Academic Press.