Effects of external acid shift and benzoate addition on cytoplasmic pH. (A) Cultures were suspended at pH 7.6 in supplemented M63 minimal media (20 mM HOMOPIPES). At time zero, 8.5 mM HCl was added to shift the external pH from pH 7.6 to 5.5. For the control, KCl (8.5 mM at pH 7.6) was added instead of HCl (B) The permeant weak acid sodium benzoate (30 mM) was added at time zero to cultures suspended at pH 7.0 in supplemented M63 minimal meida (50 mM HOMOPIPES). As a control, HOMOPIPES (pH 50 mM at 7.0) was added instead of sodium benzoate. For both experimental conditions, fluorescence was converted to pH units using the standard curves as described in Materials and Methods. Under each condition, three independent cultures were tested.