Preparation and Quantification of Mycobacterium bovis BCG lux Inoculum for Infection

Abstract

Source: Asai, M. et al., Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex. J. Vis. Exp. (2019)

This video demonstrates the preparation and quantification of Mycobacterium bovis Bacillus Calmette-Guérin lux inoculum using bioluminescence assay and the enumeration of colony-forming units. The prepared inoculum can be used for infection studies.

Protocol

NOTE: All work described below is to be carried out in a CL2 laboratory within a class 2 microbiological safety cabinet (MSC) following local health and safety guidelines.

1. Preparation of M. bovis BCG lux for Infection

1. Defrost a frozen 1.2 mL glycerol (15%) stock of M. bovis BCG lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi encoding the luciferase enzyme.
2. Inoculate 15 mL of Middlebrook 7H9 broth containing 0.2% glycerol, 10% albumin, dextrose, catalase (ADC....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5ml reaction tube (Eppendorf)	Eppendorf	22431021
20, 200 and 1000 µl pipette and filtered tips	Any supplier	n/a
24-well culture plate	Greiner	662160
25 ml pipettes and pipette boy	Any supplier	n/a
3 compartment Petri dish (94/15mm)	Greiner	637102
Centrifuge	Any supplier	n/a
Class II saftey cabinet	Any supplier	n/a
Ethanol (>99.7%)	VWR	208221.321
Glycerol	Sigma-Aldrich	G5150
Hygromycin B	Corning	30-240CR
Luminometer (Autolumat LB 953)	Berthold	34622
Luminometer tubes	Corning	352054
Microcentrifuge	Any supplier	n/a
Middlebrook 7H11 agar	BD Bioscience	283810
Middlebrook 7H9 broth	BD Bioscience	271310
Middlebrook ADC enrichment	BD Bioscience	212352
Middlebrook OADC enrichment	BD Bioscience	212240
Mycobacterium bovis BCG lux	Various	n/a
n-decyl aldehyde	Sigma-Aldrich	D7384-100G
Orbital shaking incubator	Any supplier	n/a
Phosphate-buffered saline	Sigma-Aldrich	P4417-100TAB
Polysorbate 80 (Tween-80)	Sigma-Aldrich	P8074-500ml
Erlenmeyer flask with vented cap (250 ml)	Corning	CLS40183
Winterm (V1.08)	Berthold	n/a	Program LB953.TTB