Preparation and Quantification of Mycobacterium bovis BCG lux Inoculum for Infection
Transcription
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.
