2.5. Membrane potential assay

The membrane potential of S. aureus MTCC 740 and P. aeruginosa MTCC 741 was tested using the fluorescence probe DiSC₃(5) by the method of Huang and Yousef (2014). Bacterial cells in the mid-logarithmic phase were centrifuged, washed in 5mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.8) and resuspended in the same buffer to an optical density at 600nm of 0.05. The fluorescent probe DiSC₃(5) was added to the cell suspension to a final concentration of 0.5 μM. The mixture was incubated for 15 min at room temperature to allow the uptake of the DiSC₃(5) probe. After incubation, potassium chloride was added to the cell suspension at a final concentration of 100mM. To the 900μL of cell suspension, 100 μL of 4X MIC Hydroquinone was added and incubated for 30 min and 1h separately. For the negative control, the live (untreated) bacteria were used. The proton ionophore, CCCP (10μM) treated with the bacterial pathogen for 1h was used as the positive control. A change in fluorescence due to membrane depolarization was recorded using a BD FACSCalibur flow cytometry system at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. All the experiments were performed in triplicates.