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 세스 introduce bacteria from external sources, 세스 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 세스 100 colonies 세스 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 세스 transfer 100 microliters of overnight growth 세스 a sterile microfuge tube. Next, transfer 200 microliters of each culture 세스 a 96-well plate. Remove the samples from the biosafety cabinet and centrifuge them 세스 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 세스 re-suspend the pellet. Mix the diluted bacterial strains in equal ratios 세스 create a multi-species cocktail. Transfer 50 microliters of the cocktail 세스 the conical tubes containing the sterile diet and dechorionated eggs. Be sure 세스 add the bacteria after the egg transfer 세스 prevent contamination between vials. Then place the inoculated tubes in an incubator.

To measure the Colony Forming Units or CFUs, transfer five flies 세스 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 세스 the second well containing 70 microliters of MRS. Mix the contents of the second well thoroughly. Then transfer 10 microliters from the second well 세스 the third well containing 70 microliters of MRS, and mix thoroughly. Transfer 10 microliters of each dilution 세스 an mMRS plate. Slightly incline the dish 세스 spread the dilution several millimeters down the agar’s surface and allow the liquid 세스 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 세스 100 isolated colonies. Finally, calculate the CFU per fly.