Rapid detection of respiring Escherichia coli O157:H7 in apple juice, milk, and ground beef by flow cytometry

Background: Rapid and simple methods to detect viable pathogenic microbes in foods and drinks are required. Flow cytometry was used for the rapid detection of respiring Escherichia coli O157:H7 cells in apple juice, milk, and ground beef. Methods: CTC (5-cvano-2,3-ditolyl tetrazolium chloride) was used to estimate the respiratory activity of bacteria. Fluorescein isothiocyanate (FITC)-labeled anti-E. coli O157:H7 direct antibody (FA) was used for the specific detection of target cells. Food samples were inoculated with starved E coli O157:H7 and E coli K-12 cells, and analyzed by both fluorescent microscopy and flow cytometry after double staining with FA and CTC. Results: Respiring E coli O157:H7 cells in food samples showed strong fluorescence of both FA (green) and CTC (red) thus, they could be clearly and specifically distinguished from respiring E coli K-12 or inactive cells. A good correlation was achieved in flow cytometric analysis between the numbers of inoculated viable E coli O157:H7 and those detected in milk and apple juice. The detection threshold for this flow cytometry for E coli O157:H7 in milk, apple juice, and ground beef was 10(3) cells/ml (milk and apple juice) or 10(3) cells/g (ground beet) of sample when the total bacterial number in the sample was 10(6) cells/ml. Conclusions: Respiring E coli O157:H7 in food samples can be detected specifically within a few hours. Flow cytometry with FA-CTC double staining can be used to examine food contamination with various pathogenic microbes demonstrating physiologic activity through the use of a suitable fluorescent antibody. (C) 2003 Wiley-Liss, Inc.