Measurement of the intracellular pH of Propionibacterium acnes: comparison between the fluorescent probe BCECF and 31P-NMR spectroscopy

The intracellular pH of the Gram-positive skin bacterium Propionibacterium acnes was determined using the pH-sensitive fluorescent probe 2′, 7′ bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF). The probe was introduced into the bacteria using the membrane-permeable acetoxymethyl ester BCECF-AM. The intracellular pH of the bacteria was determined by establishing a relation between the fluorescence ratio 505/450 and pH using the ionophore nigericin. To verify the intracellular pH determined using BCECF, the results were compared with those obtained using 31P-NMR spectroscopy. The effects of different external pH values and glucose addition upon the intracellular pH were examined using BCECF and 31P-NMR. Good correlation was obtained between the two techniques. Propionibacterium acnes maintained a pH gradient, inside alkaline, in the external pH range 5.0–7.4, which inverted when the pH was > 7.5. At external pH ≥ 8.5, the intracellular pH was close to the external pH. Glucose exposure did not affect the intracellular pH. Rapid, transient intracellular acidification and alkalinization brought about using NaHCO3 and NH4Cl, respectively, could be detected using BCECF. A limitation encountered when using BCECF was BCECF leakage, which could significantly affect the results if not taken into account.Key words: intracellular pH, BCECF fluorescence, 31P-NMR spectroscopy, Propionibacterium acnes.