Mycobacterium Membrane Activity Assay

Reported methods were followed to determine the membrane depolarizing and permeabilizing activities of test compounds on M. bovis BCG cultures.^{23 (link),24 (link)} Briefly, M. bovis BCG cultures at mid-log phase were diluted at OD₆₀₀ 0.1 in complete 7H9 broth. Diluted cultures were treated with test compounds at 4× MIC₉₀ for 48 h. At selected time points, aliquots were removed from culture media and tested for changes in membrane potential using BacLight Bacterial Membrane Potential kit (Life Technologies, CA) and membrane permeability using fluorescent probes SYTO-9 and propidium iodide (PI) (Molecular Probes, Invitrogen, MA). RIF at 4× MIC₉₀ (0.08 μM) was used as the negative control for both assays. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 100 μM and a detergent SDS (sodium dodecyl sulfate) at 5% (v/v) were used as the positive controls in membrane potential and permeability tests, respectively. In the membrane permeability assay, drug-treated samples were normalized against drug-free cultures (0% permeabilization) and SDS-treated cultures (100% permeabilization) to give % permeabilization.^{23 (link)}