Counting Colony Forming Units (CFUs): Quantifying Bacteria from Fly Bodies

- First, collect flies at the desired stage of development and place them in a sterile tube. To count only the internal microbiota, sterilize the exterior of the flies by submerging them in 70% ethanol. To measure both the internal and external microbiota, skip this sterilization step. Next, grind up the flies in a small volume of bacterial growth media using ceramic beads and a tissue homogenizer, or manually with a pestle. Care should be taken not to introduce bacteria from external sources, to prevent contaminating the samples.

You should now have a homogeneous mixture containing bacteria that you can culture. Bring up the volume of the homogenate using liquid bacterial medium. Perform serial dilutions of the homogenate and plate a constant volume of these dilutions on the appropriate solid growth medium for your bacteria. As the bacteria grow, colonies will form on the plate. Find the dilution that has a range of 10 to 100 colonies to determine the Colony Forming Units, or CFU, of the sample.

In the example protocol, we will inoculate sterile flies with a gnotobiotic or defined bacterial culture and collect and measure CFUs.

- Use filter tips to transfer 100 microliters of overnight growth to a sterile microfuge tube. Next, transfer 200 microliters of each culture to a 96-well plate. Remove the samples from the biosafety cabinet and centrifuge them to pellet the bacteria. After making one-, two-, and four-fold dilutions, measure the optical density or OD600 on a multi-well plate reading spectrophotometer.

After obtaining the OD600, remove the supernatant with a pipette tip and use fresh mMRS to re-suspend the pellet. Mix the diluted bacterial strains in equal ratios to create a multi-species cocktail. Transfer 50 microliters of the cocktail to the conical tubes containing the sterile diet and dechorionated eggs. Be sure to add the bacteria after the egg transfer to prevent contamination between vials. Then place the inoculated tubes in an incubator.

To measure the Colony Forming Units or CFUs, transfer five flies to a 1.7 milliliter microfuge tube containing 125 microliters of ceramic beads and 125 microliters of mMRS broth. Homogenize the flies using a tissue homogenizer. There should be no whole pieces the fly remaining. Next, dilute the homogenate with 875 microliters of mMRS, vortex the homogenate for five seconds, and pipette 120 microliters into the first well of a microtiter plate.

Remove 10 microliters from the first well and add it to the second well containing 70 microliters of MRS. Mix the contents of the second well thoroughly. Then transfer 10 microliters from the second well to the third well containing 70 microliters of MRS, and mix thoroughly. Transfer 10 microliters of each dilution to an mMRS plate. Slightly incline the dish to spread the dilution several millimeters down the agar's surface and allow the liquid to dry before moving the plate. Remove the plates from the incubator once distinct individual colonies are visible and count them from a dilution with 10 to 100 isolated colonies. Finally, calculate the CFU per fly.