This article describes a method for culturing and quantifying Mycobacterium bovis BCG lux, a genetically modified strain used in infection studies. The process involves measuring bioluminescence to assess metabolic activity and correlate it with viable bacterial counts.
Begin with a defrosted culture of Mycobacterium bovis BCG lux, a genetically modified bioluminescent strain of attenuated Mycobacterium bovis expressing the reporter luciferase enzyme.
Inoculate the suspension into a culture flask containing the antibiotic hygromycin-supplemented growth medium. Incubate. BCG lux harbors the hygromycin-resistant gene, facilitating its survival and growth.
Post-incubation, prepare suitable culture dilutions using a buffer in luminometer tubes. Transfer to the luminometer, loaded with a long-chain aliphatic aldehyde substrate solution. During the run, the aldehyde is injected into the mycobacterial suspension which enters the cells.
Bacterial luciferase catalyzes the oxidation of endogenous reduced flavin mononucleotide and the aldehyde, resulting in blue-green light emission. The measured bioluminescence intensity indicates the metabolic activity of BCG lux and correlates to the measure of viable bacteria or colony-forming units, CFU.
Next, centrifuge the remaining mycobacterial culture. Resuspend the mycobacterial pellet in a buffer containing a non-ionic surfactant to prevent mycobacterial clumping.
Using the bioluminescence measurements, dilute the suspension to obtain the desired cell density. Prepare serial dilutions of mycobacteria and plate them on hygromycin-supplemented nutrient agar plates. Incubate, allowing BCG lux to form colonies.
Calculate CFU to confirm correlation with bioluminescence intensity. The prepared BCG lux inoculum can be used for infection studies.
To begin, defrost a frozen 1.2-milliliter glycerol stock of Mycobacterium bovis BCG lux. The Montreal vaccine strain transformed with the shuttle plasmid PSMT-1 carrying the luxAB genes. In a labeled 250-milliliter Erlenmeyer flask, inoculate 15 milliliters of Middlebrook 7H9 broth with the defrosted 1.2-milliliter aliquot of BCG lux.
Place the flask in a sealed biosafety container, and incubate at 37 degrees Celcius in an orbital shaker incubator at 220 RPM for 72 hours. After incubation, prepare one to 10 dilutions of the culture in phosphate-buffered saline in luminometer tubes. Vortex, and load the luminometer tubes into the luminometer.
Load the substrate 1% n-decyl aldehyde in absolute ethanol into the injector platform and measure the bioluminescence. Convert the bioluminescence to colony-forming units to check the growth of BCG lux culture.
Transfer the culture to a centrifuge tube, and centrifuge at 2,175 times g for 10 minutes at room temperature to pellet the cells. Discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Wash the cell pellet twice in 50 milliliters of PBS-T by centrifuging at 2,175 times g for 10 minutes to prevent bacterial clumping.
Following the final wash, decant the waste supernatant, and resuspend the mycobacterial cell pellet in the required volume of PBS-T to dilute the mycobacterial suspension to the desired cell density. Then, prepare 10-fold serial dilutions of the inoculum in 24-well plates using PBS-T.
Plate out 10 microliters of each dilution onto Middlebrook 7H11 agar plates in duplicate to enumerate inoculum CFU counts.
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