Measuring Bacterial Membrane Potentials

Cell membrane potentials were determined using the fluorescence dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4 (3); Sigma, St. Louis, USA) according to methods described elsewhere [22 (link)] with minor modifications. Cells of MDR S. aureus SA-596 and MDR P. aeruginosa PA-69 were prepared as described in Section 2.6.1. Each pellet was resuspended in PBS (10⁸ CFU/mL) with one of the various concentrations of verbascoside (0, MIC, or 2 × MIC). Then, the samples were incubated for 30 min at 30 °C. A bacterial suspension (200 μL) and the fluorescent probe DiBAC4 (3) (1 mM final concentration) were added to each well of a black, opaque 96-well plate (Corning, NY, USA). The mixtures were then incubated at 37 °C for 30 min in the dark. Cell suspensions were analysed using a fluorescence spectrophotometer (BioTek, Winooski, VT, USA) with excitation and emission wavelengths of 488 nm and 518 nm, respectively. Changes in the fluorescence intensity of DiBAC4 (3) were recorded to monitor the membrane potential. All the values were corrected for cell number and background fluorescence.