Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva

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.