Rapid Detection and Identification of Microbes in Food

Improved methods for the microbiological analysis of foods are needed, especially quantitative methods. More efficient ways to enumerate microbial cells are essential for gathering data on incidence, exposure, and infective dose, for use in quantitative risk assessment strategies to enhance food safety. The objectives of this project included testing of molecular probes for specific enumeration of bacteria by direct epifluorescence microscopy and comparison to conventional standard methods of enumeration in food systems for sensitivity and accuracy. A fluorescent oligonucleotide probe complementary to a published 16S ribosomal RNA sequence of Escherichia coli was tested in situ in hybridizations on membrane filters. The oligonucleotide-direct epifluorescent filter technique (oligo-DEFT) was developed and involved a 2-hour hybridization with the probe after membrane filtration of water, beverages and sprouts homogenates. Enumeration was performed by semi-automated counting of cells by microscopy and image analysis. The probe showed reactivity to several genera of the Enterobacteriaceae, and was not specific for E. coli. Heat-killing of E. coli cells at 60 C did not destroy the ability of the probe to hybridize to the cells. Natural coliform populations in aquarium water were countable after a 90 minute incubation of the filter on nutrient agar ('resuscitation') before the hybridization step. Natural coliforms in sprouts were directly countable without the resuscitation step. Oligo-DEFT counts were correlated with standard coliform counts by membrane filtration or plating with conventional selective media.