title: Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.
abstract: BACKGROUND
  Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment.
  
  
  METHODS
  We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively.
  
  
  CONCLUSIONS
  With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.
authors.affiliation: Department of Biology, General Botany, University of Milan, Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Barbesti
authors.forename: S
authors.initials: S
authors.lastname: Citterio
authors.forename: M
authors.initials: M
authors.lastname: Labra
authors.forename: M D
authors.initials: MD
authors.lastname: Baroni
authors.forename: M G
authors.initials: MG
authors.lastname: Neri
authors.forename: S
authors.initials: S
authors.lastname: Sgorbati
mesh_terms: D000818:Animals; D001412:Bacillus subtilis / Q000254:growth & development / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D052160:Benzothiazoles; D003959:Diamines; D004783:Environmental Microbiology; D004926:Escherichia coli / Q000254:growth & development / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D009930:Organic Chemicals; D011419:Propidium / Q000378:metabolism; D011804:Quinolines; D011817:Rabbits; D013194:Staining and Labeling
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D011419:Propidium