-- views • 3:40 min
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.
Place a subset of the infected flies into individual microcentrifuge tubes on ice. Add 250 microliters of PBS to each tube, and use a pestle to homogenize the flies. Next, plate the homogenate on an LB agar plate using a spiral plater.
Alternatively, to plate the homogenate by serial dilution, transfer each fly homogenate to the first row of a 96-well plate, and fill each well of the remaining rows, with 90 microliters of PBS. Using a multichannel pipette, take 10 microliters from the first row containing fly homogenate, and dispense the homogenate into the second row.
Pipette up and down at least 10 times to thoroughly mix the homogenate, then, take 10 microliters of the homogenate, and transfer it to the next row. Repeat this procedure using the remaining rows. Starting from the bottom row, use the multichannel pipette to take 10 microliters from each well, and deposit it on an LB plate.
Ensure that the samples are dispensed as discrete spots that do not touch each other. Repeat this step until all wells have been sampled from each row, dispensing them in descending order of dilution on the LB plate. Whichever plating method is used, take care not to overgrow the plates so colonies remain small and discrete.
Remove the plate from the incubator once the experimental bacteria have grown visible colonies but before colonies from the Drosophila gut microbiota become visible approximately 24 hours later.
For spiral plates, count the colonies that grow using an automated colony counter that can estimate the bacterial load per milliliter of homogenate, based on the number and position of the colonies on the plate.
For spot plates, manually count the colonies for each fly from whichever dilution contains 30 to 300 colonies, and calculate the number of bacteria per milliliter of original homogenate.