Intracellular ph determination of pristinamycin-producing Streptomyces pristinaespiralis by image analysis

Intracellular ph (pH(i)) is an essential parameter in the regulation of int racellular processes. Thus, its measurement might provide clues regarding t he physiological state of cells cultivated in vitro, pH(i) of the filamento us, pristinamycin-producing Streptomyces pristinaespiralis was determined b y epifluorescence microscopy and image analysis using the pH-sensitive fluo rescent probe BCECF-AM [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. Staining cell culture samples (OD660 = 1) of S. prist inaespiralis with 20 muM BCECF at 28 degreesC for 30 min yielded a green/re d fluorescence ratio (R-527/600) that correlated with the pH(i) of the cell s for values ranging from 6.5 to 8.5. When S. pristinaespiralis was cultiva ted in pristinamycin-producing conditions (in batch mode, with a constant e xternal ph of 6.8), the measured pH(i) varied between 6.3 and 8.7. In fact, pH(i) correlated with the excretion of pristinamycins and glucose consumpt ion during the production process.