A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.