The development of a rapid method for measuring intracellular pH (pH) in single bacterial cells is described. subsp. and were used as test organisms. The method is based upon fluorescence microscopy and ratio imaging of cells stained with carboxyfluorescein succinimidyl ester. After staining, the bacteria were immobilized on a membrane filter and transferred to a closed perfusion chamber, allowing control of the cell environment during analysis. The set-up was optimized with regard to the use of neutral-density filters and background subtraction, for determining the excitation ratio 490 nm/435 nm (R) independent of the excitation light intensity, and to reduce photobleaching. This allowed for time-lapse studies with multiple exposures. To study the pH of subsp. and in response to different extracellular pH (pH) values, an calibration curve was constructed in the pH range 5·0–8·5. Distinct differences between the two cultures were observed. maintained a near-neutral pH almost independently of pH (5·0–8·0), whereas the pH of subsp. decreased with decreasing pH. In pure and mixed cultures at pH 5·0, the pH values of subsp. and were 6·1 ± 0·2 and 7·5 ± 0·2, respectively. This difference in pH may explain how subsp. obtains a competitive advantage over at low pH.


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