Preparation and Quantification of Mycobacterium bovis BCG lux Inoculum for Infection

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.