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).

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