Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth

Take a multi-well plate containing serial dilutions of the test compound, an aminoglycoside antibiotic.

Add a suspension of Mycobacterium tuberculosis — a pathogenic bacterium — into the wells and incubate.

In the cytoplasm, the test compound binds to the mycobacterial 30S ribosomal subunit, inhibiting normal protein synthesis and eventually leading to mycobacterial death.

Post-incubation, add resazurin — a weakly-fluorescent water-soluble dye, to the cultures. Incubate.

Inside viable metabolically active mycobacteria, dehydrogenase enzymes reduce blue-colored resazurin to a pink-colored fluorescent resorufin.

Using a fluorescence plate reader, measure the resorufin fluorescence intensity in the wells.

A low fluorescence intensity value with increasing test compound concentrations suggests its cytotoxic effects against the mycobacteria.

To carry out a resazurin assay, grow M. tuberculosis in 7H9ADST to mid-log phase. Dilute the culture with 7H9ADST to an OD₆₀₀ of 0.01. Use 7H9ADST to dilute the compounds to two times the testing concentrations, and aliquot 100 microliters of each diluted compound into each well of a clear 96-well plate.

Transfer 100 microliters of the diluted bacte