Membrane Potential Measurement of S. mutans

The membrane potential of untreated and EGCG-treated planktonic S. mutans was measured using the cationic dye 3,3′-diethyloxacarbocyanine iodide (DiOC₂(3); Molecular Probes, Eugene, OR, USA) by flow cytometry according to the manufacturer’s instructions. DiOC₂(3) exhibits green fluorescence in all bacterial cells, but the fluorescence shifts toward red emission at higher membrane potential values. Briefly, an overnight culture of S. mutans was resuspended in PBS to an OD of 0.3. The bacterial suspension was divided into 1 ml aliquots with different concentrations of EGCG and stained with 10 µl of 3 mM DiOC₂(3) for 30 min in the dark. The samples were analyzed by flow cytometry (LSR-Fortessa flow cytometer, BD Biosciences), using the 488 nm excitation laser and collecting the data using the green (530nm) and red (610/620 nm) filters [40 (link)]. In addition, we measured the forward scatter (FSC) and the side scatter (SSC) of the untreated and EGCG-treated bacteria. FSC detects light scatter along the path of the laser and its intensity is proportional to the diameter of the cell. SSC measures light scatter at a ninety-degree angle relative to the laser and provides information about the granularity of the cell.