Assessing Cell Layer Permeability in Non-Replicating Persistent Mycobacterium tuberculosis

Begin with tubes containing the non-replicating persistent, or NRP stage of Mycobacterium tuberculosis, which has a thickened outer layer.

Add glass beads to one tube and shake it to mechanically disrupt the mycobacteria’s outer layer, enhancing permeability. 

Next, transfer the bead-beaten mycobacterial suspension to a fresh tube.

Add fluorescently-labeled rifampicin, an anti-tuberculosis antibiotic, to both the untreated and bead-beaten cell suspensions.

Incubate with shaking to allow fluorescently-labeled rifampicin to enter the mycobacteria.

Over time, more rifampicin enters the mycobacteria.

Centrifuge the samples to separate the cells and remove the supernatant containing unbound rifampicin. 

Resuspend the mycobacteria in fresh media.

Analyze the samples using flow cytometry. As cells pass through a laser beam, they emit fluorescence proportional to the intracellular fluorescent-rifampicin.

At different time points, untreated NRP cells show lower fluorescence than bead-beaten cells.

This indicates that the thickened outer layer restricts rifampicin entry, supporting bacterial persistence during the host–pathogen interaction.