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

Abstract

A fluorescent oligonucleotide probe complementary to a published 16S ribosomal RNA sequence ofEscherichia coli 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 hybridization on intact cells collected on a membrane filter surface (‘oligo-DEFT’), instead of the traditional acridine orange staining. The filter was examined by epifluorescence microscopy, and digital image analysis was used for the cell enumeration. Oligo-DEFT counts were linear over the range of 103–106cells ml−1and correlated with DEFT counts (r=0·99). The oligo-DEFT detection limit was approximately 1 cell ml−1. Oligo-DEFT counts correlated with standard coliform counts by membrane filtration or plating on selective media for analysis of water and beverages artificially inoculated with E. coli. Heat inactivation of the cells did not destroy their reactivity in the oligo-DEFT. Natural coliform populations in aquarium water were countable in the oligo-DEFT after a 90-min incubation of the filter on nutrient agar before the hybridization step, which allowed the dormant cells to increase their rRNA content. Natural populations in bean sprouts were countable directly, without having to perform the incubation before hybridization.