Colony Forming Units (CFUs) tellen: bacteriën uit vlieglichamen kwantificeren

- 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 over