Germany) was added to a final concentration of 10 mg/L, and accumulation measured with and without the addition of 100 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), an electron transport chain uncoupler that inhibits energy-dependent efflux. Following addition of ciprofloxacin, 0.5 mL samples were removed at 15 s intervals for 1 min, at 30 s intervals for up to 5 min, then at 10, 15, 20, 30, 45, 60, 90 and 120 min intervals. The fluorescence of the supernatant was determined with a Luminescence Spectrometer LS-50B (Perkin Elmer, USA) at excitation and emission wavelengths of 275 and 440 nm, respectively. The concentration of ciprofloxacin in the supernatant was calculated by comparison with a standard curve for ciprofloxacin (0.01–0.250 mg/L) in 0.1 M glycine hydrochloride (pH 3). Accumulation of ciprofloxacin (ng/mg cells) was calculated by subtracting the absorbed ciprofloxacin at time zero from the amount of ciprofloxacin eluted at various time intervals. The experiments were performed in duplicate on two separate occasions in order to ensure reproducibility. The accepted standard deviation for all accumulation results was 10% with respect to the mean values taken at the respective sampling times