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

1. Setting Up Replicating and Non-replicating MIC₉₀ Assays

1. Inoculate M. tuberculosis, M. bovis BCG or M. smegmatis at an OD₅₈₀ of 0.01-0.1 and expand to mid-log phase (OD₅₈₀ ~0.5) in Middlebrook 7H9-ADN (Middlebrook 7H9 containing 0.2% glycerol, 0.2% dextrose, 0.5% albumin, and 0.085% NaCl) or 7H9-OADC (Middlebrook 7H9 containing 0.2% glycerol and 10% OADC supplement).
2. Grow M. tuberculosis and M. bovis BCG as ~20 ml standing cultures in cell culture flasks and M. smegmatis with shaking in polypropylene round-bottom (4 ml culture) or 50 ml conical centrifuge tubes (10-20 ml culture). Incubate pathogenic mycobacteria at 37 °C with 20% O₂ and 5% CO₂, and M. smegmatis at 37 °C with 20% O₂.
3. Set up a minimal-inhibitory concentration (MIC90)-style experiment under replicating and non-replicating conditions (Figure 1).
   NOTE: Test agents are usually assayed in duplicates or quadruplicates to permit testing 4, or 2 compounds, respectively, per 96-well microplate. For example, in a 96-well plate, one test agent can be assayed in rows A-D and another in rows E-H. The DMSO (vehicle) is in columns 1, 2, and 12, and the test agent dilution series runs from column 3 (lowest concentration) to column 11 (highest concentration). MIC90-style assays typically employ a 2-fold dilution series. There are numerous non-replicating models available for mycobacteria and for illustrative purposes, we are using a multi-stress model of non-replication.
   1. For the replicating assay, distribute 200 μl cells in 7H9-ADN at an OD₅₈₀ of 0.01 into all wells of a clear-bottomed, tissue culture-treated 96-well plate.
   2. For the non-replicating assay, wash cells twice in phosphate-buffered saline (PBS) containing 0.02% tyloxapol, and resuspend cells in non-replicating medium (0.05% KH₂PO₄, 0.05% MgSO₄, 0.005% ferric ammonium citrate, 0.0001% ZnCl₂, 0.1% NH₄Cl, 0.5% BSA, 0.085% NaCl, 0.02% tyloxapol, 0.05% butyrate; pH adjusted to 5.0 with 2 N NaOH).
      1. Dilute cells to an OD₅₈₀ of 0.1 in a non-replicating medium and add NaNO₂ from a freshly prepared 1 M stock to a final concentration of 0.5 mM.
      2. Distribute 200 μl cells at an OD₅₈₀ of 0.1 into all wells of a clear-bottomed, tissue culture-treated 96-well plate.
         NOTE: Prepare compound dilutions as 100-fold stock solutions in DMSO. Thus, a typical MIC plate testing the impact of a molecule at a final concentration of 0.4-100 μg/ml would require stock solutions of 0.04-10 mg/ml in DMSO.
   3. Add 2 μl of dilutions of test agent 1 into rows A-E and 2 μl of dilutions of test agent 2 into rows E-H. Mix thoroughly.
   4. Add 2 μl vehicle control (usually DMSO) into control wells, columns 1, 2, 12 (rows A-H). Mix thoroughly.
   5. For each experiment, include at least one positive control such as rifampicin from 0.004 to 1 μg/ml (replicating assay) and/or 0.08 to 20 μg/ml (non-replicating assay).
      NOTE: The use of 6-bromo-1H-indazol-3-amine9 at 0.1 to 25 μg/ml is recommended as a control compound that has selective, NaNO2-dependent activity in the multi-stress model of non-replication.
   6. For replicating assays, incubate microplates at 37 °C at 20% O₂ and 5% CO₂ for 7 days (M. tuberculosis and M. bovis BCG) or 1-48 hr (M. smegmatis). For the multi-stress model of non-replication, incubate microplates for 7 days at 37 °C at 1% O₂ and 5% CO₂ (M. tuberculosis and M. bovis BCG).

2. Inoculation of CARA Microplates

1. At time points in which the CARA will be used as a read-out, carefully resuspend well contents of the MIC90-style assay plate using a p200 multichannel pipette set at 50-75 μl. Pipette up and down at least 5-10 times and gently swirl the well contents in a circular motion using the pipette tips.
2. Transfer 10 μl of assay well contents to the CARA microplate. Ensure that the order of the well contents on the assay plate matches the order of the well contents of the CARA microplate. Avoid splashing during the transfers and make sure the 10 μl are spotted into the middle of the CARA microplate wells. Confirm the 10 μl absorbs into the CARA microplate.
   NOTE: There are no dilutions required prior to spotting cells on the CARA microplates.
3. Bind stacks of CARA microplates with plate tape and then place them into a resealable plastic bag. Incubate CARA microplates at 37 °C with 20% O₂ (M. smegmatis) or 1% O₂ and 5% CO₂ (M. tuberculosis and M. bovis BCG).
4. For M. tuberculosis and M. bovis BCG, replicating assays: incubate for 7 days; for M. tuberculosis and M. bovis BCG non-replicating assays, incubate for 10 days; for M. smegmatis, replicating assays, incubate for 1-2 days; for M. smegmatis, non-replicating assays, incubate 2-3 days.
   NOTE: Times are estimates and may be modified accordingly for different replicating, non-replicating, and stress conditions.

3. Developing CARA Microplates

1. Develop the CARA microplates when a film of bacterial growth, or larger, macroscopic colonies, are visible on the negative (vehicle) control wells.
   NOTE: After prolonged incubation, CARA microplate wells often appear dry and we recommend pre-wetting well contents with sterile PBS. This serves to prevent the charcoal from absorbing resazurin, which can lead to low fluorescence or well-to-well variation.
2. Using a single set of 12 p200 tips with a multichannel pipette, dispense 40 µl of sterile PBS along the side of the wells and allow the PBS to distribute across the top of the agar/bacterial microcolonies.
3. Prepare CARA developing reagent by mixing 5 mg resazurin (0.01% final) and 50 ml of 5% Tween-80 in PBS. Vortex and sterile filter.
   NOTE: An alternative CARA developing reagent can be prepared by mixing commercially prepared resazurin liquid solution at 1:1 (vol/vol) with 10% Tween80 in PBS.
4. Add 50 µl of freshly prepared CARA developing reagent to each well of the CARA microplate using a 12-channel pipette. Rock plates back and forth a few times to help distribute reagents across the agar and bacterial mat in each well.
5. Place the plates in a resealable plastic bag and incubate at 37 °C for at least 30 min for M. smegmatis and 45-60 min for M. tuberculosis or M. bovis BCG.
   NOTE: If the vehicle control wells fail to turn pink within the first hour, the plates can be re-bagged and incubated for longer periods of time.
6. Prior to reading fluorescence, place CARA microplates in a biosafety hood for 15 min at room temperature with their lids removed. When using BSL3 spectrophotometers outside of a biosafety cabinet, adhere an optical quality PCR sticker over the plate and seal tightly by pressing gently on the sticker surface with a soft paper towel.
7. Determine fluorescence via top read with excitation at 530 nm and emission at 590 nm. It is not necessary to blank the plate.