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The development of a rapid method for measuring intracellular pH (pHi) in single bacterial cells is described. Lactobacillus delbrueckii subsp. bulgaricus and Listeria innocua 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 (R490/435) independent of the excitation light intensity, and to reduce photobleaching. This allowed for time-lapse studies with multiple exposures. To study the pHi of Lb. delbrueckii subsp. bulgaricus and L. innocua in response to different extracellular pH (pHex) values, an in vivo calibration curve was constructed in the pHi range 5.0-8.5. Distinct differences between the two cultures were observed. L. innocua maintained a near-neutral pHi almost independently of pHex (5.0-8.0), whereas the pHi of Lb. delbrueckii subsp. bulgaricus decreased with decreasing pHex. In pure and mixed cultures at pHex 5.0, the pHi values of Lb. delbrueckii subsp. bulgaricus and L. innocua were 6.1 +/- 0.2 and 7.5 +/- 0.2, respectively. This difference in pHi may explain how Lb. delbrueckii subsp. bulgaricus obtains a competitive advantage over L. innocua at low pHex.

Use of fluorescence ratio imaging for intracellular pH determination of individual bacterial cells in mixed cultures