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

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.

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