title: Rapid, absolute, and simultaneous quantification of specific pathogenic strain and total bacterial cells using an ultrasensitive dual-color flow cytometer.
abstract: 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 O157:H7 and harmless E. coli DH5alpha was used as a model system. Pathogenic E. coli O157: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 O157:H7 emits both red and green fluorescence, while E. coli DH5alpha 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 DH5alpha (R(2) = 0.9998). Bacterial cell mixtures with varying proportions of E. coli O157:H7 and E. coli DH5alpha were measured with good ratio correspondence. We applied the established approach to detecting artificially contaminated drinking water samples; E. coli O157:H7 of 1.0 x 10(2) 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.
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Yang
authors.forename: Lina
authors.initials: L
authors.lastname: Wu
authors.forename: Shaobin
authors.initials: S
authors.lastname: Zhu
authors.forename: Yao
authors.initials: Y
authors.lastname: Long
authors.forename: Wei
authors.initials: W
authors.lastname: Hang
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Yan
mesh_terms: D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000378:metabolism; D004926:Escherichia coli / Q000302:isolation & purification*; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry
chemical_list: D000906:Antibodies; D000941:Antigens; D005456:Fluorescent Dyes