Rapid, Absolute, and Simultaneous Quantification of Specific Pathogenic Strain and Total Bacterial Cells Using an Ultrasensitive Dual-Color Flow Cytometer

This paper describes a rapid and sensitive strategy for the absolute and simultaneous quantification of specific pathogenic strain and total bacterial cells in a mixture. A laboratory-built compact, high-sensitivity, dual channel flow cytometer (HSDCFCM) was modified to enable dual fluorescence detection. A bacterial cell mixture comprising heat-killed pathogenic Escherichia coli E. coli 0 157: H7 and harmless E. coli DH5 alpha was used as a model system. Pathogenic E. coli 0 157:H7 cells were selectively labeled by red fluorescent probe via antibody-antigen interaction, and all bacterial cells were stained with membrane-permeable nucleic acid dye that fluoresces green. When each individual bacterium passes through the interrogating laser beam, E. coli 0 157:117 emits both red and green fluorescence, while E. coli DH5 alpha. exhibits only green fluorescence. Because the fluorescence burst generated from each individual bacterial cell was easily distinguished from the background, accurate enumeration and consequently absolute quantification were achieved for both pathogenic and total bacterial cells. By using this strategy, accurate counting of bacteria at a density above 1.0 x 10(5) cells/mL can be accomplished with 1 min of data acquisition time after fluorescent staining. Excellent correlation between the concentrations measured by the HSDCFCM and the conventional plate-counting method were obtained for pure-cultured E. coli O157:H7 (R-2 = 0.9993) and E. coli DH5 alpha (R-2 = 0.9998). Bacterial cell mixtures with varying proportions of E. coli O157:H7 and E. coli DH5 alpha were measured with good ratio correspondence. We applied the established approach to detecting artificially contaminated drinking water samples; E. coli 0157:H7 of 1.0 x 101 cells/mL were accurately quantified upon sample enrichment. It is believed that the proposed method will find wide applications in many fields demanding bacterial identification and quantification.