This study investigates the cytotoxic effects of an aminoglycoside antibiotic on Mycobacterium tuberculosis using a resazurin assay. The method involves measuring fluorescence intensity to assess bacterial viability following treatment with the antibiotic.
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 bacterial suspension into each well. Incubate the plates at 37 degrees Celsius in a humidified incubator for 5 days. Dissolve 10 milligrams of resazurin in 100 milliliters of deionized water, and filter-sterilize the solution. Add 30 microliters of resazurin solution to the wells. Then, incubate the cells for 48 hours.
Bacterial growth is indicated by a color conversion from blue to pink. Carry out quantitative analysis according to the text protocol.
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