Intracellular pH measurement

S. aureus cells were collected and washed with 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 8.0), and the samples were then mixed with 3 μM carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) probes and incubated gently at 37 °C for 30 min in the dark. To remove the nonconjugation effect, the samples were eluted with 10 mM glucose and washed twice with HEPES buffer. The fluorescence density was then measured using a fluorescence microplate detector (Bio-Tek, USA) with excitation wavelengths of 440 and 490 nm and an emission wavelength of 520 nm. The standard curve was determined using a series of pH buffers (50 mM glycine, 50 mM citric acid, 50 mM NaHPO₄^.2H₂O, and 50 mM KCl; pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), and the fluorescence intensity (FI) ratios (FI_{Ex=440 nm, Em=520 nm} to FI_{Ex=490 nm, Em=520 nm}) were recorded.