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Applied and Environmental Microbiology, April 2002, p. 2031-2035, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.2031-2035.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Rapid Assessment of the Physiological Status of the Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102 by Flow Cytometry

Yoshinori Hiraoka1 and Kazuhide Kimbara1,2*

Department of Built Environment, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8502,1 Environmental Biotechnology Laboratory, Railway Technical Research Institute, Kokubunji, Tokyo 185-8540, Japan2

Received 1 October 2001/ Accepted 9 January 2002

The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R2 = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = -0.9776 x + 98.36; R2 = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (<=1 h) and distinguishes correctly among live, dead, and permeabilized cells.

* Corresponding author. Mailing address: Environmental Biotechnology Laboratory, Railway Technical Research Institute, 2-8-38 Hikaricho, Kokubunji, Tokyo 185-8540, Japan. Phone: 81-42-573-7316. Fax: 81-42-573-7349. E-mail: kimbara{at}rtri.or.jp.

Applied and Environmental Microbiology, April 2002, p. 2031-2035, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.2031-2035.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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