This study investigates the interaction between pathogenic bacteria and Drosophila melanogaster, focusing on the mechanisms of bacterial ingestion and colonization. The methodology includes a series of controlled experiments to assess bacterial viability post-infection.
Take a vial with a lid that holds nutrient agar overlaid with filter paper.
Apply a suspension of pathogenic bacteria onto the filter paper.
Introduce a starved Drosophila melanogaster fly into the vial, then place the lid. Incubate to allow feeding and ensure bacterial ingestion.
The ingested bacteria enter the gut and penetrate the protective membrane lining the lumen.
The bacteria adhere to the underlying epithelium, release toxins that promote epithelial damage and invasion, and colonize internal tissues.
Immerse the fly in alcohol to remove surface-associated bacteria, then rinse to remove alcohol.
Add a buffer and homogenize the fly to release the internal bacterial contents.
Perform serial dilutions of the homogenate in a microplate. Next, plate the dilutions onto nutrient agar and incubate to allow colony formation.
Count the colonies to determine the number of viable bacterial cells resulting from oral infection in the fly.
Two to four hours before the oral infection, transfer the flies to standard agar vials containing no food. Meanwhile, for each fly, prepare an infection vial.
First, load the lid of a 7 milliliter sample tube with 500 microliters of standard sugar agar and let it solidify. Next, stick a disk of filter paper in the agar. Then pipette 100 microliters of bacterial culture directly onto the filter disc. For controls, use 5% sucrose water.
After the starvation period, transfer single flies to the prepared tubes. After incubating the flies for 18 to 24 hours, surface-sterilize the flies. Load individuals into a tube containing 100 microliters of 70% ethanol.
After 20 to 30 seconds, remove the ethanol and replace it with 100 microliters of triple-distilled water. After another 20 to 30 seconds, remove the water. Then add 100 microliters of PBS and homogenize the fly.
Transfer the homogenate to the top row of a 96-well plate and add 90 microliters of 1x PBS to every well below. Then, using 10 microliter aliquots, serially dilute this sample to distinguish a range of CFU values. Next, plate the serial dilutions on an LB nutrient agar plate in 5 microliter droplets.
Position the droplets so they are discreetly located on the plate. Then incubate the plate overnight. The next day, calculate the number of CFUs from the dilution that produces 10 to 60 clearly visible colonies.
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