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1. Setting Up Replicating and Non-replicating MIC90 Assays
<ol>
	<li>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).</li>
	<li>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 &#176;C with 20% O2 and 5% CO2, and M. smegmatis at 37 &#176;C with 20% O2.</li>
	<li>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.
	<ol>
		<li>For the replicating assay, distribute 200 &#956;l cells in 7H9-ADN at an OD580 of 0.01 into all wells of a clear-bottomed, tissue culture-treated 96-well plate.</li>
		<li>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).
		<ol>
			<li>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.</li>
			<li>Distribute 200 &#956;l 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 &#956;g/ml would require stock solutions of 0.04-10 mg/ml in DMSO.</li>
		</ol>
		</li>
		<li>Add 2 &#956;l of dilutions of test agent 1 into rows A-E and 2 &#956;l of dilutions of test agent 2 into rows E-H. Mix thoroughly.</li>
		<li>Add 2 &#956;l vehicle control (usually DMSO) into control wells, columns 1, 2, 12 (rows A-H). Mix thoroughly.</li>
		<li>For each experiment, include at least one positive control such as rifampicin from 0.004 to 1 &#956;g/ml (replicating assay) and/or 0.08 to 20 &#956;g/ml (non-replicating assay). 
		NOTE: The use of 6-bromo-1H-indazol-3-amine9 at 0.1 to 25 &#956;g/ml is recommended as a control compound that has selective, NaNO2-dependent activity in the multi-stress model of non-replication.</li>
		<li>For replicating assays, incubate microplates at 37 &#176;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 &#176;C at 1% O2 and 5% CO2 (M. tuberculosis and M. bovis BCG).</li>
	</ol>
	</li>
</ol>
2. Inoculation of CARA Microplates
<ol>
	<li>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 &#956;l. Pipette up and down at least 5-10 times and gently swirl the well contents in a circular motion using the pipette tips.</li>
	<li>Transfer 10 &#956;l 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 &#956;l are spotted into the middle of the CARA microplate wells. Confirm the 10 &#956;l absorbs into the CARA microplate. 
	NOTE: There are no dilutions required prior to spotting cells on the CARA microplates.</li>
	<li>Bind stacks of CARA microplates with plate tape and then place them into a resealable plastic bag. Incubate CARA microplates at 37 &#176;C with 20% O2 (M. smegmatis) or 1% O2 and 5% CO2 (M. tuberculosis and M. bovis BCG).</li>
	<li>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.</li>
</ol>
3. Developing CARA Microplates
<ol>
	<li>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.</li>
	<li>Using a single set of 12 p200 tips with a multichannel pipette, dispense 40 &#181;l of sterile PBS along the side of the wells and allow the PBS to distribute across the top of the agar/bacterial microcolonies.</li>
	<li>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.</li>
	<li>Add 50 &#181;l 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.</li>
	<li>Place the plates in a resealable plastic bag and incubate at 37 &#176;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.</li>
	<li>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.</li>
	<li>Determine fluorescence via top read with excitation at 530 nm and emission at 590 nm. It is not necessary to blank the plate.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Middlebrook 7H9</td>
			<td>Beckton Dickinson</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H11</td>
			<td>Beckton Dickinson</td>
			<td>298810</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook OADC</td>
			<td>Beckton Dickinson</td>
			<td>212351</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, heat shock</td>
			<td>Roche</td>
			<td>3118958001</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated charcoal</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, Dulbecco&#39;s Ca2+ and Mg2+ free</td>
			<td>Life Technologies / Invitrogen</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma</td>
			<td>P8074</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Sigma</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr>
			<td>6-bromo-1H-indazol-3-amine</td>
			<td>Alfa Aesar</td>
			<td>H34095</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate, heptahydrate</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate, heptahydrate</td>
			<td>Sigma</td>
			<td>Z0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Butyric acid, liquid</td>
			<td>Sigma</td>
			<td>B103500</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin powder</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>J.T. Baker</td>
			<td>4058-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepared resazurin solution</td>
			<td>Invitrogen</td>
			<td>DAL1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Molecular Devices</td>
			<td>M5</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well, tissue culture treated microplates</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoirs</td>
			<td>VWR</td>
			<td>89094-678</td>
			<td></td>
		</tr>
		<tr>
			<td>Resealable plastic bags</td>
			<td>VWR</td>
			<td>395-94602</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL Polypropylene round-bottom tubes</td>
			<td>Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 Cell culture flask</td>
			<td>Corning</td>
			<td>431464U</td>
			<td></td>
		</tr>
	</tbody>
</table>

a charcoal agar resazurin assay to evaluate test compound activity against mycobacteria

Source: Gold, B. et al., <a href="https://app.jove.com/v/54690">Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/21915/21915_Figure1.jpg" /> 
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.

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

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 ...

Take a multi-well plate containing Mycobacteria. Add increasing test compound concentrations to the wells.
Incubate. Depending on the compound&#39;s action on Mycobacteria, it can either inhibit mycobacterial growth, displaying a bacteriostatic activity, or eliminate Mycobacteria, demonstrating bactericidal activity.
Transfer well aliquots to a charcoal agar resazurin assay, or CARA, microplate containing agar with activated charcoal to absorb the test compound, preventing carryover effects.
Incubate. The compound&#39;s bacteriostatic activity inhibits mycobacterial replication, while bactericidal activity reduces mycobacterial viability.
Add buffer, resazurin, and a non-ionic surfactant to disperse resazurin in the wells.&#160;&#160;
In the metabolically active Mycobacteria, the dehydrogenases reduce the blue-colored, weakly fluorescent resazurin to a pink, fluorescent resorufin.
Using a microplate reader, measure the resorufin fluorescence in the wells.
Wells lacking resorufin fluorescence indicates the compound&#39;s bactericidal activity.
Varying fluorescence intensity across wells indicates the live bacterial load, reflecting the concentration-dependent bactericidal or bacteriostatic potency of the test compounds.

a-charcoal-agar-resazurin-assay-to-evaluate-test-compound-activity

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

80 Aufrufe. Quelle: Gold, B. et al., Visualisierung des Aktivkohle-Agar-Resazurin-Assays für die semiquantitative Zählung von Mykobakterien mit mittlerem Durchsatz. J. Vis. Exp. (2016) Dieses Video zeigt den Charcoal Agar Resazurin Assay (CARA) zur Bewertung der Aktivität von antimikrobiellen Verbindungen gegen Mycobacterium tuberculosis. CARA hilft bei der Bewertung der Wirksamkeit dieser Verbindungen, indem es ihren Einfluss auf das Bakterienwachstum misst und zwischen bakteriziden und bakteriostatisc...

Serie: Ein Aktivkohle-Agar-Resazurin-Assay zur Bewertung der Aktivität der Testsubstanz gegen Mykobakterien - Konzept

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

System zur Wirksamkeit und Zytotoxizität Screening von Inhibitoren Targeting intrazellulärer<em&gt; Mycobacterium tuberculosis</em

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Forschung

JoVE Journal

Immunologie und Infektion

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Ein Hochdurchsatz-Assay unterstützte Wirksamkeit von Medikamenten gegen Makrophagen agierten zur Bewertung<em&gt; Mycobacterium tuberculosis</em

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Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Verwendung der wirbellosen Galleria mellonella als Infektionsmodell zur Untersuchung des Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

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

Transkript

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