A Charcoal Agar Resazurin Assay to Evaluate Test Compound Activity Against Mycobacteria
Published: January 31, 2024
Abstract
Source: Gold, B. et al., Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria. J. Vis. Exp. (2016)
This video showcases the Charcoal Agar Resazurin Assay (CARA) for evaluating the activity of antimicrobial compounds against Mycobacterium tuberculosis. CARA aids in assessing the efficacy of these compounds by gauging their impact on bacterial growth and distinguishing between bactericidal and bacteriostatic effects.
Protocol
1. Setting Up Replicating and Non-replicating MIC90 Assays 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). Grow M. tuberculosis and M. bovis BCG…
Representative Results
Figure 1: Schematic of the broth MIC90 assay and CARA with anticipated results. Both MIC90 and CARA results are presented for 8 possible activities. The color-coding for MIC90 microplate wells is white (no growth) and brown (growth), and for CARA microplates is black (no fluorescence) and pink (resorufin fluorescence). Data are hypothetical.
Disclosures
The authors have nothing to disclose.
Materials
| Middlebrook 7H9 | Beckton Dickinson | 271310 | |
| Middlebrook 7H11 | Beckton Dickinson | 298810 | |
| Middlebrook OADC | Beckton Dickinson | 212351 | |
| BSA, heat shock | Roche | 3118958001 | |
| Activated charcoal | Sigma | ||
| PBS, Dulbecco's Ca2+ and Mg2+ free | Life Technologies / Invitrogen | 14190-144 | |
| Tween 80 | Sigma | P8074 | |
| Rifampicin | Sigma | R3501 | |
| 6-bromo-1H-indazol-3-amine | Alfa Aesar | H34095 | |
| Potassium phosphate monobasic | Sigma | P0662 | |
| Magnesium sulfate, heptahydrate | Sigma | M1880 | |
| Ferric ammonium citrate | Sigma | F5879 | |
| Zinc sulfate, heptahydrate | Sigma | Z0251 | |
| Ammonium chloride | Sigma | A9434 | |
| Butyric acid, liquid | Sigma | B103500 | |
| Resazurin powder | Sigma | R7017 | |
| Sodium chloride | J.T. Baker | 4058-01 | |
| Prepared resazurin solution | Invitrogen | DAL1100 | |
| Spectrophotometer | Molecular Devices | M5 | |
| 96-well, tissue culture treated microplates | Corning | 3595 | |
| Reagent reservoirs | VWR | 89094-678 | |
| Resealable plastic bags | VWR | 395-94602 | |
| 14 mL Polypropylene round-bottom tubes | Corning | 352059 | |
| 50 mL conical centrifuge tube | Corning | 352070 | |
| 75 cm2 Cell culture flask | Corning | 431464U |
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Cite This Article
A Charcoal Agar Resazurin Assay to Evaluate Test Compound Activity Against Mycobacteria. J. Vis. Exp. (Pending Publication), e21915, doi: (2024).