A Charcoal Agar Resazurin Assay to Evaluate Test Compound Activity Against Mycobacteria

Take a multi-well plate containing Mycobacteria. Add increasing test compound concentrations to the wells.

Incubate. Depending on the compound's action on Mycobacteria, it can either inhibit mycobacterial growth, displaying a bacteriostatic activity, or eliminate Mycobacteria, demonstrating bactericidal activity.

Transfer well aliquots to a charcoal agar resazurin assay, or CARA, microplate containing agar with activated charcoal to absorb the test compound, preventing carryover effects.

Incubate. The compound's bacteriostatic activity inhibits mycobacterial replication, while bactericidal activity reduces mycobacterial viability.

Add buffer, resazurin, and a non-ionic surfactant to disperse resazurin in the wells.  

In the metabolically active Mycobacteria, the dehydrogenases reduce the blue-colored, weakly fluorescent resazurin to a pink, fluorescent resorufin.

Using a microplate reader, measure the resorufin fluorescence in the wells.

Wells lacking resorufin fluorescence indicates the compound's bactericidal activity.

Varying fluorescence intensity across wells indicates the live bacterial load, reflecting the concentration-dependent bactericidal or bacteriostatic potency of the test compounds.