Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth

Overview

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 1 mL of deionized water; the final concentration is 50 mg/mL. Filter-sterilize and store at -20 °C. Add 0.5 mL of kanamycin stock solution per 1 L of 7H9ADST.
   NOTE: This medium should be made fresh, so scale the volumes appropriately according to the culture size.
4. Grow M. tuberculosis in 7H9ADST supplemented with kanamycin in standing culture. Shake the culture daily and dilute it before the OD₆₀₀ reaches 1.0 to avoid clumping.
   NOTE: The M. tuberculosis strain used for the development of this method was H37Rv transformed with pJAK2.A plasmid. pJAK2.A is an integrative plasmid based on the pMV361 vector, which allows high-level expression of the firefly luciferase gene from the hsp60 promoter and can be selected using kanamycin.

2. In-broth Activity Analysis Using a Resazurin Assay

1. Grow M. tuberculosis in 7H9ADST to the mid-log phase (~ 0.5 - 0.8 OD600). Dilute the culture with the 7H9ADST to 0.01 OD₆₀₀. Dilute the compounds in 7H9ADST to 2x the testing concentrations and aliquot 100 µL of each diluted compound into each well.
2. Transfer 100 µL of the diluted bacterial suspension into each well. Allow the plates to incubate at 37 °C in a humidified incubator for 5 days. Dissolve 10 mg of resazurin in 100 mL of deionized water and a sterile filter.
3. Add 30 µL of resazurin solution and monitor the color change after 48 h; bacterial growth is indicated by a color conversion from blue to pink.
   NOTE: A quantitative analysis can also be performed by measuring either the fluorescence at 590 nm with excitation at 530 - 560 nm or the absorbance at 570 nm and 600 nm.

Materials

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