Quantification of Colony-Forming Units of Gut-Associated Bacteria in Drosophila Flies

Begin with a microplate containing homogenates prepared from Drosophila flies. These flies harbored a controlled population of a specific bacterium in their gut.

The homogenates contain lysed fly tissues and released gut bacteria.

Aspirate the suspended bacteria and serially dilute across multiple microplates.

Spot small volumes from each dilution onto agar plates, ensuring that the droplets remain separated.

Incubate to allow bacteria to proliferate into visible colonies.

Capture images of the plates to quantify colony-forming units (CFUs), which represent the number of viable bacterial cells capable of forming individual colonies.

Using image analysis software, align each image using a reference line, define a boundary, and apply a uniform grid to segment it into counting zones, each corresponding to a single spot.

Adjust the brightness threshold to distinguish individual colonies from the background, then run the analysis to count colonies in each spot and quantify the CFUs.

Place three rectangular MRS agar growth plates with their lids open in the biosafety cabinet to dry for at least 10 minutes. Prepare two dilution plates by adding 100 microliters of PBS to each well of a sterile 96-