Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

The overall goal of the CARA is to provide a medium-throughput semi-quantitative assessment of the activity of compounds against replicating and non-replicating mycobacteria. The main advantage of this technique is that as a CFU surrogate, the CARA allows one to rapidly test the dose and time dependency of a compound. This method can help answer key questions in the tuberculosis drug discovery field such as determining if a compound is bacteriostatic or bactericidal against replicating bacteria and if it is bactericidal against non-replicating bacteria.

Demonstrating the method will be Julia Roberts, an associate in our laboratory. Add 450 milliliters of water to a one liter glass beaker or 900 milliliters of water to a two liter Erlenmeyer flask. Include an autoclavable stir bar in the beaker or flask.

Next, add a Middlebrook 7H11 powder, activated charcoal to a 0.4%final concentration and 50%glycerol solution to achieve a 0.2%final concentration. Cover the opening of the flask or beaker with aluminum foil and attach to glass with autoclave tape. After autoclaving the medium, cool it to approximately 55 to 65 degrees Celsius on a magnetic stir plate set to low speed to maintain the charcoal in suspension.

While working aseptically in a biosafety hood that has a magnetic stir plate, remove the foil from the medium and add 100 or 50 milliliters of OADC supplement to the two liter flask or one liter beaker respectively and continue to mix. Using a multichannel pipette with eight or 12 filter tips, fill a 96-well microplate with 200 microliter well of 7H11-OADC-charcoal from the reagent reservoir or beaker, working quickly to avoid agar solidification and the formation of bubbles. Place stacks of CARA microplates in resealable plastic bags to avoid drying out and store them at four degrees Celsius.

Inoculate polypropylene round-bottom or conical centrifuge tubes containing four 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 re-suspend the cells. Use non-replicating medium to dilute the cells to an OD580 of 0.1 and add sodium nitrate to a final concentration of 0.5 to 5 millimolar from a freshly prepared one 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 two microliters of test agent one into rows A through E and two microliters of test agent two 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 one to 48 hours.

At the time points in which the CARA will be used as a read-out, use a p200 multichannel pipette set at 50 to 75 microliters to carefully re-suspend the well contents of the MIC90-style assay plate by pipetting up and down five 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, ensuring at the 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 five milligrams of resazurin and 50 milliliters of 5%Tween80 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. It is not necessary to blank the plate. To analyze the data, plot the inhibitor concentration on the X-axis as a log10 scale and fluorescence on the Y-axis on a linear scale.

Refer to the text protocol for additional details. The concentration of test agent that results in failure of CARA fluorescence to rise above background levels is the CARA-MBC greater than equal to 99 which is shown here. The great than equal to 99 subscript indicates that the CARA-MBC provides an estimated concentration of test agent, that gives rise to a greater than equal to two log10 bacterial kill.

Compounds are suspected of exerting a post antibiotic effect if they display a static window, defined as a greater than four-fold shift to the right between the MIC and CARA curves. The static window indicates that a molecule active against replicating M.tuberculosis may be bacteriostatic instead of bactericidal. For molecules with a potent post antibiotic effect, static windows may be difficult to observe and they're apparent only after inspection of an expanded Y-axis for CARA fluorescence as seen here.

Replicating and non-replicating active molecules tested by MIC90 and CARA are shown here. While isoniazid and linezolid appear to have activity against non-replicating bacteria by the MIC90 assay, the CARA suggest that they are inactive under the non-replicating conditions tested. In this CARA experiment, exposing M.smegmatis to increasing concentrations of rifampicin revealed an impact in as little as one hour and displayed increasing bactericidal activity between three and 24 hours killing greater than equal to two to three log10 at approximately 10 micrograms per milliliter by 24 hours.

Once mastered, this technique can be done in approximately two hours to prepare plates, under an hour to transfer cells to CARA plates, and approximately one to two hours to develop and measure fluorescence of CARA plates if it is done properly. While attempting this procedure, it's important to remember to use the CARA as a predictive assay to determine if a compound has bactericidal or bacteriostatic activity against mycobacteria that are replicating or non-replicating. After watching this video, you should have a good understanding of how to set up a CARA and analyze data in a manner that helps predict the type of activity of a compound.

Thanks for watching and good luck with your experiments.