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

Inoculate polypropylene round-bottom or conical centrifuge tubes containing 4 or 20 milliliters respectively of Middlebrook 7H9-ADN, or 7H9-OADC with M. smegmatis at an OD₅₈₀ of 0.01 to 0.1. Incubate the M. smegmatis cultures at 37 degrees Celsius and 20% O₂ with shaking, expanding the cultures to mid-log phase or an approximate OD₅₈₀ of 0.5.

To set up a minimal inhibitory concentration style experiment under replicating and non-replicating conditions, distribute 200 microliters of cells in 7H9-ADN at an OD₅₈₀ of 0.01 into all the wells of a clear-bottomed tissue culture-treated 96-well plate.

For the non-replicating assay, use PBS containing 0.02% tyloxapol to wash the cells twice and use non-replicating medium to resuspend the cells. Use non-replicating medium to dilute the cells to an OD₅₈₀ of 0.1, and add sodium nitrite to a final concentration of 0.5 to 5 millimolar from a freshly prepared 1 molar stock.

Distribute 200 microliters of cells at an OD₅₈₀ of 0.1 into all wells of a clear-bottomed tissue culture-treated 96-well plate. After preparing test agents according to the text protocol, add 2 microliters of test agent #1 into rows A through E, and 2 microliters of test agent #2 into rows E through H, and mix thoroughly.

For replicating assays, incubate M. smegmatis microplates at 37 degrees Celsius, 20% O₂, and 5% CO₂ for 1 to 48 hours. At the time points in which the CARA will be used as a readout, use a P200 multichannel pipette set at 50 to 75 microliters to carefully resuspend the well contents of the MIC90-style assay plate by pipetting up and down 5 to 10 times. Then, use the pipette tips to gently swirl the contents of the wells in a circular motion.

Transfer 10 microliters of the undiluted assay well contents to the corresponding wells of the CARA microplate, avoiding splashing during transfers, and ensuring that the 10-microliter aliquot is spotted into the middle of the CARA microplate wells. With plate tape, bind stacks of CARA microplates, and then, place them into a resealable plastic bag. Incubate the M. smegmatis CARA microplates at 37 degrees Celsius with 20% O₂ for one to two days for replicating plates and for two to three days for non-replicating plates.

When a film of bacterial growth, or larger macroscopic colonies are visible in the negative control wells, use a single set of 12 P200 tips with a multichannel pipette to dispense 40 microliters of sterile PBS along the side of the wells, and allow the PBS to distribute across the top of the agar bacterial microcolonies.

Prepare CARA developing reagent by mixing 5 milligrams of resazurin and 50 milliliters of 5% Tween-80 in PBS. Add 50 microliters of freshly prepared CARA developing reagent to each well of the CARA microplate. Then, rock the plates back and forth a few times to help distribute the reagent across the agar and bacterial mat in each well. Place the plates into a resealable plastic bag, and incubate at 37 degrees Celsius for at least 30 minutes for M. smegmatis.

Prior to reading fluorescence, place CARA microplates in a biosafety hood for 15 minutes at room temperature with the lids removed. When using BSL3 spectrophotometers outside of a biosafety cabinet, adhere an optical quality PCR sticker over the plate, and use a soft paper towel to seal tightly by pressing gently on the sticker surface. Determine the fluorescence via top read with excitation at 530 nanometers and emission at 590 nanometers.