Sensitive detection of Escherichia coli O157 : H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

Rapid, direct methods are needed to assess active Bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respir atory activity, using the pathogen Escherichia coli O157:H7, Counterstainin g with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteri a by epifluorescence microscopy. Broth-grown E, coli O157:H7 was used to in oculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inocu lated meat was diluted and homogenized in a stomacher and then incubated wi th paramagnetic beads coated with anti-O157 specific antibody. After IMS, c ells with magnetic beads attached were stained with CTC and then an anti-O1 57 antibody-fluorescein isothiocyanate conjugate and filtered for microscop ic enumeration or solid-phase laser cytometry, Enumeration by laser scannin g permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liqui d sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-n egative plate counts in the inoculum were as follows: intercept = 1.06, slo pe = 0.89, and r(2) = 0.95 (n = 13). The corresponding results for inoculat ed peptone were as follows: intercept = 0.67, slope = 0.88, and r(2) = 0.98 (n = 24). Recovery of target bacteria on heads by the IMS-CTC-FAb method,c ompared with recovery by sorbitol MacConkey agar plating, yielded greater n umbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, withi n 5 to 7 h, the IMS-CTC-FAb method detected greater numbers off. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.