A pH-sensitive fluorescent dye, 2′, 7′-bis-(2-carboxyethyl)-5 and 6-carboxyfluorescein (BCECF), was used to determine intracellular pH (pHin). 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 pHin determination. In the presence of glucose, however, the fluorescence decreased by 57%. Thus, the pHin 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 pHin was almost equal to the extracellular pH (pHout) for pHout values of between 8 and 5, indicating that protons permeated easily across the S. mutans cell membrane. For pHout between 5 and 4, pHin was constant at around 5, suggesting that the cell membrane was impermeable to protons, or that a cytoplasmic buffering system functioned, pHin decreased at pHout values of < 4. The constant pHin at acidic pHout levels could protect intracellular components, such as proteins, against acidification by sugar fermentation.
ASJC Scopus subject areas