Method for Assessment of Viability and Morphological Changes of Bacteria in the Early Stage of Colony Formation on a Simulated Natural Environment

ABSTRACT A quantitative analysis of changes in the physiological status of bacterial cells is a fundamental type of study in microbiological research. We devised a method for measuring the viability of bacteria in the early stage of colony formation on a simulated natural environment. In this method, a solid medium containing soil extract was used, and the formation of bacterial microcolonies on a membrane filter was determined by use of a laser scanning cytometer combined with live-dead fluorescent dyes. A polychlorinated biphenyl degrader, Comamonas testosteroni TK102, was used in this study. Surprisingly, approximately 20% of the microcolonies had their growth stopped and eventually died. In the presence of biphenyl, the growth arrest was increased to 50%, and filamentous cells were observed in the colonies. Predicted intermediate metabolites of biphenyl were added to the medium to determine the relationship between the change of viability and the production of metabolites, and the addition of 2,3-dihydroxybiphenyl showed low viability. The arrest was not observed to occur on nutrient-rich medium, suggesting that the change in viability might occur in a nutrient-poor natural condition. The results of this study demonstrated that toxic metabolites of xenobiotics might change cell viability in the natural environment.

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