This study investigates the shedding of pathogenic bacteria by Drosophila flies infected with a gut-colonizing bacterial pathogen. The methodology involves transferring infected flies to sterile environments and measuring bacterial colonies to assess transmission potential.
Begin with a Drosophila fly previously infected by a gut-colonizing bacterial pathogen.
Transfer the fly into a sterile tube containing a fly food medium.
Leave the fly undisturbed to feed on the food.
Inside the fly gut, the pathogenic bacteria utilize the nutrients from the food and multiply.
When the fly excretes, some bacteria are also released.
After a fixed period, transfer the fly into a fresh tube with food and repeat the process multiple times.
Then, discard the fly from the last tube.
Add a buffer to each tube and vortex to release bacteria from the fly excreta.
Prepare serial dilutions of the recovered solution from each tube.
Spot the diluted solutions onto a nutrient agar plate and incubate to allow bacterial colonies to grow.
Count the colonies to determine the number of viable bacteria shed by the infected fly over time, which reflects its potential to transmit infection.
To measure bacterial shedding, transfer single flies to 1.5 milliliter tubes containing about 50 microliters of Lewis medium.
Change the tube every 24 hours. After three tubes have been populated for 24 hours, dispose of the flies. After each transfer, measure the bacteria contained in the tube.
Mix the tube content with 100 microliters of PBS using a vortex. Then measure the CFUs in the mixture by plating serial dilutions.
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