Intracellular and extracellular pHs of Streptococcus mutans after addition of acids: Loading and efflux of a fluorescent pH indicator in streptococcal cells

A pH-sensitive fluorescent dye, 2′, 7′-bis-(2-carboxyethyl)-5 and 6-carboxyfluorescein (BCECF), was used to determine intracellular pH (pH_in). The efflux of BCECF loaded into oral streptococcal cells was determined after incubation of the cells at 35°C for 20 min in the presence and absence of glucose. In the absence of glucose, the fluorescence of intracellular BCECF in Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius and Streptococcus sobrinus decreased only very slightly, indicating that the dye could be useful for pH_in determination. In the presence of glucose, however, the fluorescence decreased by 57%. Thus, the pH_in of S. mutans cells was measured by the BCECF method in the absence of glucose at various acidic pH levels by adding lactic, acetic and hydrochloric acids to the cell suspensions. The pH_in was almost equal to the extracellular pH (pH_out) for pH_out values of between 8 and 5, indicating that protons permeated easily across the S. mutans cell membrane. For pH_out between 5 and 4, pH_in was constant at around 5, suggesting that the cell membrane was impermeable to protons, or that a cytoplasmic buffering system functioned, pH_in decreased at pH_out values of < 4. The constant pH_in at acidic pH_out levels could protect intracellular components, such as proteins, against acidification by sugar fermentation.