Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth

Abstract

Source: Zheng, X., et al. System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis. J. Vis. Exp. (2017)

This video demonstrates an in-broth assay to determine antibiotic efficacy against Mycobacterium tuberculosis. The bacterial culture is incubated with different concentrations of a bactericidal antibiotic. The bactericidal efficacy is measured by assessing the presence of viable bacteria using a resazurin assay.

Protocol

1. Bacterial Strain and Growth Medium

1. Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of dextrose, and 4.05 g of sodium chloride in 460 mL of deionized water. Filter-sterilize the ADS and store it at 4 °C.
2. Make 7H9 broth by adding 4.7 g of 7H9 powder and 2 mL of glycerol to 900 mL of purified water. Autoclave the 7H9 broth at 121 °C for 10 min and allow it to cool to room temperature before proceeding. Make 7H9ADST by adding 100 mL of ADS and 0.5 mL of Tween80 to 900 mL of 7H9 broth. Store at 4 °C.
3. Weigh 50 mg of kanamycin sulfate and dissolve it in....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Middlebrook 7H9	Becton, Dickinson and Company	271210
Tween80	Fisher Scientific	T164
Albumin, Bovine pH7	Affymetrix	10857
Dextrose	Fisher Scientific	BP350
Sodium Chloride	Fisher Scientific	BP358
kanamycin sulfate	Fisher Scientific	BP906
Resazurin	Alfa Aesar	B21187
Glycerol	Fisher Scientific	BP229
M. tuberculosis H37Rv
96-well flat bottom white plate	Corning	3917
95-well flat bottom clear plate	Corning	3595
Transparent plate sealer	Thermo Fisher Scientific	AB-0580
Luminometer	Applied Biosystems	Tropix TR717