Flow cytometry for multiplex detection of plant pathogenic bacteria, direct viable counting and cell sorting

Flow cytometry (FCM) allows multiparameter analysis and quantification of particles based on size, granularity and on emission of fluorescent light, after staining with a fluorescent dye. We developed a simple FCM immunodetection procedures for different plant pathogenic bacteria, including Ralstoni...

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Bibliographic Details
Published in:Phytopathology 2004-06, Vol.94 (6)
Main Authors: Van der Wolf, JM, Peters, J, van der Zouwen, PS, van den Bulk, RW, Tebaldi, N D, Bergervoet, JHW
Format: Article
Language:English
Subjects:
Clavibacter michiganensis
Ralstonia solanacearum
Xanthomonas axonopodis
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Summary:Flow cytometry (FCM) allows multiparameter analysis and quantification of particles based on size, granularity and on emission of fluorescent light, after staining with a fluorescent dye. We developed a simple FCM immunodetection procedures for different plant pathogenic bacteria, including Ralstonia solanacearum (Rsol), Clavibacter michiganensis subsp. sepedonicus (Cms) and Xanthomonas axonopodis pv. phaseoli. Plant extracts were filtered and stained with fluorochrome conjugated antibodies. The entire procedure was completed within I h and allowed detection of >10.000 cells per ml of plant extract. Detection levels were improved 10 times by enrichment of bacteria in semi-selective media prior to FCM. Staining of Cms with Alexa488 conjugated antibodies (green) and of Rsol with R-phycoerythrins conjugated antibodies (red) permitted detection of both pathogens simultaneously. FCM was also evaluated for direct viable counting of bacterial cells using combinations of propidium iodide (PI, red). SYTO9 (green) and Calcein-AM (green after conversion). SYTO9 penetrates all cells. PI only (dead) cells with damaged membranes, whereas calceinAM is only converted by viable cells. Combination of PI and Alexa488-labelled antibodies allowed detection of the viable cells of a specific pathogen in mixtures of bacteria. FCM-based cell sorting offers possibilities for verification through isolation by dilution plating and PCR-amplification.
ISSN:0031-949X