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

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.