A RAPID, DIRECT METHOD FOR ENUMERATING RESPIRING ENTEROHEMORRHAGIC ESCHERICHIA-COLI O157-H7 IN WATER

Simple, rapid methods for the detection and enumeration of specific ba cteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory a ctivity with a modified fluorescent-antibody (FA) technique, for the e numeration of specific viable bacteria. Bacteria in suspensions were c aptured by filtration on nonfluorescent polycarbonate membranes that w ere then incubated on absorbent pads saturated with CTC medium. A spec ific antibody conjugated with fluorescein isothiocyanate was reacted w ith the cells on the membrane filter. The membrane filters were mounte d for examination by epifluorescence microscopy with optical filters d esigned to permit concurrent visualization of fluorescent red-orange C TC-formazan crystals in respiring cells which were also stained with t he specific FA. Experiments with Escherichia coli O157:H7 indicated th at both respiratory activity and specific FA staining could be detecte d in logarithmic- or stationary-phase cultures, as well as in cells su spended in M9 medium or reverse-osmosis water. Following incubation wi thout 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 ce lls compared with R2A agar plate counts gave a strong linear regressio n (R = 0.997). Differences in injury did not appear to affect CTC redu ction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiell a pneumoniae.