Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.
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.
Barbesti
S
S
Department of Biology, General Botany, University of Milan, Milan, Italy.
Citterio
S
S
Labra
M
M
Baroni
M D
MD
Neri
M G
MG
Sgorbati
S
S
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
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D011419:Propidium