PI Membrane Permeability Assay

Cell membrane integrity is associated with cell morphology and antibiotic susceptibility. Propidium iodide (PI) is considered to only stain cells with irreparably damaged membranes (Yang et al., 2015). Therefore, the cell wall and membrane integrity of M. tuberculosis H37Ra after dicumarol treatment were checked using a PI membrane permeability assay.

Briefly, the log phase of M. tuberculosis H37Ra was added with dicumarol (2×MIC) in a flask for 24 h, while untreated M. tuberculosis H37Ra was used as a control. The bacteria were harvested by centrifugation at 3,000 × g for 10 min and washed with 0.9% NaCl solution. Then, the bacteria were stained in 250 μl of a 0.9% NaCl solution that included 2 μl of the PI dye (Sigma) for 15 min in the dark. After staining, the bacteria were centrifuged and washed twice with 0.9% NaCl solution. The pellets were resuspended in 0.9% NaCl solution. The bacteria with damaged membranes were stained with PI and visualized as a red color under fluorescence microscopy, while the intact bacterial cells were not stained.