Intracellular pH of halobacteria can be determined by the fluorescent dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein.

Determination of the internal pH of halobacterial cells grown in 4M salt solution has proven to be a difficult problem. We now report the steady state cytosolic pH of Halobacterium halobium S-9 to be 7.2. Intracellular pH was determined after the cells were loaded with the membrane permeable precursor of the pH sensitive dye 2', 7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein-acetyxymethyl ester - (BCECF/AM). In order to minimize light-scattering in the measurement of the fluorescence, a thin cuvette was newly devised. This method should be suitable for studies of the cytosolic pH in other bacteria.