Preparation and Quantification of Mycobacterium bovis BCG lux Inoculum for Infection

Overview

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) enrichment, and 50 µg/mL hygromycin with a defrosted 1.2 mL aliquot of BCG lux, in a labeled 250 mL Erlenmeyer flask.
3. Place the flask in a sealed biosafety container and incubate at 37 °C in an orbital shaker incubator at 220 rpm for 72 h (or until the culture reaches the mid-log phase of growth).
4. Check the growth of BCG lux culture by preparing 1:10 dilutions of the culture in luminometer tubes using phosphate-buffered saline (PBS, pH 7.4, 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride) in duplicate. Vortex, and load the luminometer tubes into the luminometer and measure the bioluminescence (relative light unit [RLU]/mL) using n-decyl aldehyde as the substrate (1% v/v in absolute ethanol).
   NOTE: The ratio of RLU/colony forming units (CFU) was previously determined to be 3:1 when BCG lux was grown in vitro in Middlebrook 7H9 broth.
5. Centrifuge the culture at 2,175 x g for 10 min at room temperature to pellet the cells and discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Dispose of all culture waste with disinfectants appropriate for mycobacteria following local guidelines.
6. Wash the cell pellet twice in PBS containing 0.05% polysorbate 80 (PBS-T) to prevent bacterial clumping.
7. Following the final wash, decant waste supernatant, resuspend the mycobacterial cell pellet in PBS-T, and dilute the mycobacterial suspension to the desired cell density using RLU measurements.
8. Prepare ten-fold serial dilutions of the inoculum in 24-well plates using PBS-T. Plate out 10 µL onto Middlebrook 7H11 agar plates (0.5% glycerol, 50 µg/mL hygromycin, 10% oleic acid, albumin, dextrose, catalase [OADC]) in duplicate to enumerate inoculum CFU counts.

Materials

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