This article details a method for measuring the in vivo bacterial burden of Bacillus Calmette-Guérin (BCG) in insect larvae using bioluminescence. The process involves homogenizing infected larvae, measuring bioluminescence, and counting colony-forming units to assess bacterial viability.
To measure the in vivo bacterial burden, begin with sterilized insect larvae pre-infected with Bacillus Calmette-Guérin, or BCG lux. BCG lux is a genetically modified Mycobacterium bovis BCG strain expressing luciferase — a bioluminescence-producing enzyme.
Transfer an insect larva into a buffer-containing lysing matrix tube with steel balls. Using a homogenizer, homogenize the larva, releasing the intracellular components, including bacteria, into the buffer.
Centrifuge the tube to collect the homogenate and transfer it to a luminometer tube.
Wash the matrix tube with a detergent-containing buffer to solubilize cellular fragments and release residual bacteria.
Combine the fractions in the same luminometer tube and add the luciferase substrate. Substrates enter into bacteria and interact with luciferase enzymes, resulting in bioluminescence. Load the tube into a luminometer and measure the bioluminescence.
The luminometer measures the intensity of emitted light and calculates the relative light unit, or RLU — indicative of the in vivo burden of BCG in an insect larva.
Spread the diluted homogenate suspension on a piperacillin-containing agar plate and incubate to selectively grow BCG as distinct colonies. The colonies on the plate represent the colony-forming unit, or CFU. The RLU to CFU ratio correlates bioluminescence with bacterial viability.
Every 24 hours, randomly select five infected larvae, and use a cotton bud swab soaked in 70% ethanol to gently sterilize the larval surfaces. Place the larvae individually into two-milliliter lysing matrix tubes containing 800 microliters of sterile PBS. Then, use a homogenizer to homogenize the larvae for 60 seconds at six meters per second.
Centrifuge the lysing tubes at 3,500 times g for five seconds to remove the homogenate from the lids, and carefully decant the homogenate into five sterile luminometer tubes individually. Reserve 100 microliters of homogenate in a sterile 1.5-milliliter reaction tube. Recover any remaining homogenate by washing the lysing matrix tubes with one milliliter of PBST, and pipette into the corresponding luminometer tubes.
Vortex the luminometer tubes, and measure the bioluminescence of the homogenates on a luminometer. Then, prepare 10-fold serial dilutions of the reserved 100 microliters of homogenate in 24-well culture plates using PBST. Pipette 10 microliters of the dilution onto each Middlebrook 7H11 agar plate, and use a sterile plate spreader to spread. Incubate at 37 degrees Celsius for two weeks.
After that, count colony-forming units.
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