Ethidium Bromide Uptake Assay in M. tuberculosis

Ethidium bromide uptake was measured as previously described (27 (link)). M. tuberculosis mc²7000 and derivative strains were grown to mid-exponential phase (OD₆₀₀ of 0.4 to 0.6), pelleted by centrifugation, washed once with PBS-T, and resuspended in PBS-T to an OD₆₀₀ of 0.4 to 0.5. Ethidium bromide was added at a 2-μg/ml final concentration, and uptake was measured using black, flat-bottom, 96-well microplates (Corning) and a Synergy H1 Hybrid plate reader (BioTek) in top-reading mode with excitation at 544 nm and emission at 590 nm. Uptake rates were determined using data in the linear range between 0 and 30 min and are the mean values ± standard deviations of at least three independent experiments.