4.6. Membrane Fluidity

Membrane fluidity was monitored by measuring fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH; Life Technologies) as previously reported with minor exceptions [31]. Briefly, logarithmic phase S. aureus ATCC 6538 cells were washed and resuspended in 0.85% sterile saline at a cell density of 1 × 10⁹ CFU/mL. The bacterial suspensions were incubated with different concentrations of CHQA at 25 °C for 1 h. Then, 1 × 10⁻⁶ M DPH was added to the above mixture, followed by further incubation for 30 min in the dark. Fluorescence polarization measurements were carried out on a Multi-Mode microplate reader (Synergy H1, Biotek Co., Winooski, VT, USA) equipped with 360/40 nm fluorescence excitation filter and 460/40 nm fluorescence emission filter. Suspensions of unlabeled cells with the same concentration served as reference blanks to subtract the excitant light scattering and other nonspecific contributions to the fluorescence signal. The polarization value is calculated by the following formula:

where I_VV and I_VH are the fluorescence intensities emitted in the vertical and horizontal directions when the excitation beam is oriented vertically, respectively, and G is the grating factor.