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. 2002 Apr;68(4):2031-5.
doi: 10.1128/AEM.68.4.2031-2035.2002.

Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry

Yoshinori Hiraoka  1 , Kazuhide Kimbara
Affiliations

Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry

Yoshinori Hiraoka et al. Appl Environ Microbiol. 2002 Apr.

Abstract

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; R(2) = 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; R(2) = 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 (<or=1 h) and distinguishes correctly among live, dead, and permeabilized cells.

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Figures

FIG. 1.
FIG. 1.
Histograms of live (open histogram) and ethanol-fixed (closed histogram) cells of C. testosteroni TK102 after CAM staining. Exponential-phase cells were washed in PBS (A) or HEPES buffer (B) before CAM staining.
FIG. 2.
FIG. 2.
Histograms of ethanol-fixed (closed histogram) and live (open histogram) cells of C. testosteroni TK102 after PI staining.
FIG. 3.
FIG. 3.
Dual-parameter histograms of exponential-phase (A) and ethanol-fixed (B) cells stained with CAM and PI. About 96% of exponential-phase cells were in Q4 (live cells) (A), and 100% of ethanol-fixed cells were in Q1 (dead cells) (B). Signals in Q2 were stained with both CAM and PI (permeabilized cells). Nonfluorescent debris (signals in Q3) was excluded.
FIG. 4.
FIG. 4.
Fluorescence micrographs of C. testosteroni TK102 after staining with CAM and PI. Live (A), dead (B), and permeabilized cells (C) fluoresced green, red, and both green and red, respectively.
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