3.7. Cytoplasmic Membrane Fluidity Assay

Laurdan dye was used to assess membrane fluidity as previously described using S. aureus ATCC 25923 as a model organism [28,29,37]. To conduct the assay, bacteria were grown overnight in LB and sub-cultured 1:100 in LB supplemented 1.25 mM CaCl₂, 0.5 mM MgCl₂, and 0.2% glucose. The bacteria were grown to mid-log phase (OD600 0.3–0.6). Bacteria were adjusted to an OD600 of 0.4 and stained with 10 μM Laurdan dye (in 1% DMSO) for 10 min. Opaque black half area 96-well plates (Costar 3694) were prepared containing 2× compounds in 50 μL supplemented PBS (1.25 mM CaCl₂, 0.5 mM MgCl₂ and 0.2% glucose) by adding 1 μL of 100× compounds in DMSO. Compound plates were warmed to 30 °C. The Laurdan-treated cells were washed 3–4 times in pre-warmed supplemented PBS and 50 μL per well was added to the prepared compound plate. In parallel, a compound plate was prepared containing compounds and buffer only. The bacteria plate was used to subtract the background signal from compounds in solution before data analysis. Fluorescence measurements were taken over the course of 5 min using a Perkin Elmer 2102 EnVision Multilabel plate reader (EX 340/EM 440 and 510 nm).

General polarization (GP) of the emission signal was calculated using the following formula Equation (4):

where I₄₄₀ is the fluorescence emission intensity at 440 nm and I₅₁₀ is the fluorescence emission intensity at 510 nm. The GP increases from baseline as the membrane rigidity increases and decreases as the fluidity increases.