Measuring E. coli Bacterial Load in Drosophila melanogaster following E. coli Infection

Begin with adult Drosophila melanogaster flies injected with E. coli. At desired time points post-injection, transfer anesthetized flies into separate microcentrifuge tubes on ice, reducing their metabolic activity.

Add buffer and homogenize the flies, breaking down the tissue, releasing E. coli and endogenous gut microbiota.

Transfer each E. coli-containing fly homogenate to multi-well plate wells. Fill the remaining wells with buffer. Perform serial dilution of the E. coli-containing homogenate.

Deposit the diluted homogenate suspension from each well as discrete spots on a nutrient agar plate, starting from the lowest dilution. Allow the spots to absorb into the agar. Incubate the plate overnight to enable E. coli multiplication, forming visible colonies. The incubation duration is critical to prevent the formation of the fly's endogenous gut microbiota colonies.

Count the E. coli colonies for each fly from the dilutions with countable non-overlapping colonies. Calculate the colony-forming unit — the viable bacterial load — in the starting homogenate at different time points post-injection.

Decreased E. coli load over time suggests bacterial clearance by the fly's immune system.