The overall goal of this procedure is to rear fruit flies that are free of associated microorganisms. This method can help any researcher in the Drosophila field control for, manipulate, or study the microbiota, including its relationship to various host traits. The main advantage of this technique is that it permits researchers to rear fruit flies in the complete absence of external microorganisms.
Generally, individuals new to this method will struggle, because the sterilization and rinsing steps must be conducted without introducing any contaminating microbes. To begin the experiment, streak previously prepared cultures on modified MRS, or mMRS, agar plates. Incubate the plates overnight at 30 degrees Celsius.
Incubate anaerobic cultures in an airtight container, and flood it with carbon dioxide before sealing. The next day, pick a single colony from the mMRS plate into a test tube containing mMRS broth. Grow lactobacillus species under static conditions, and grow acetobacter species with shaking.
Prepare the sterile diet in a one-liter Erlenmeyer flask. Microwave the diet until it has boiled three sequential times, and mix the flask by swirling in between each boil. Transfer the diet to a wide-mouth container on a stir plate to maintain stirring, and transfer the diet to conical centrifuge tubes.
Loosely cap the tubes and place them in a covered autoclavable polypropylene rack. Autoclave the fly diet. When the autoclave is complete, remove the racks and immediately shake each rack horizontally to ensure the diet does not separate.
Allow the diet to cool on a shaker for exactly 45 minutes. After cooling, immediately shake the diet horizontally by hand. Microwave the grape juice agar ingredients.
Bring the mixture to a boil three times, and add 10 grams of frozen grape juice concentrate to increase egg visibility on the agar plate. When the agar has cooled to 55 degrees Celsius, pour 20 milliliters into 100-millimeter Petri dishes, and allow the agar to solidify. Next, pour the yeast paste onto the agar plate, and make sure that the surface is covered, then pour off the excess, leaving a thin yeast residue behind.
Transfer the agar plates to the bottom of a cage. Transfer 250 flies into the container, and cover it with a lid. Cover the mesh-protected hole with an empty Petri dish lid.
Incubate the flies. Place nylon mesh into a plastic bushing to prepare the sieve for egg collection. If the same flies will be used the next day, immediately transfer them to a new cage containing freshly yeasted grape juice agar plates.
The grape juice plate in the previous cage contains the fruit fly eggs. Remove dead flies with a clean paint brush, and take caution not to break up the agar. Rinse the agar plate with distilled watar, and gently brush the eggs from the agar surface.
Then pour the slurry over the mesh to collect the eggs. Sterilize the biosafety cabinet with 70%ethanol, then turn on the UV light. Spray all non-biological supplies with 70%ethanol, and immediately place them in the biosafety cabinet, then sterilize them with UV light.
It is critical to ensure that good aseptic technique is practiced when working within, or when adding or removing new materials to the biosafety cabinet. Transfer the bushing with the eggs into a 120-milliliter specimen cup, and slowly pour 90 milliliters of 0.6%sodium hypochlorite solution or 7%bleach into the bushing just below the rim. Using forceps, periodically move the bushing up and down in the solution to re-suspend the eggs.
Transfer the bushing directly into a second specimen cup pre-filled with 90 milliliters of bleach. At the end of the second bleach treatment, the eggs should begin to adhere to the sides of the bushing. Discard the bleach and completely rinse the bushing with sterile water.
Again, use forceps to re-suspend the eggs several times during each wash. By the end of the third wash, most eggs should be attached to the side of the bushing. Using a sterile paint brush, transfer the eggs from the side of the bushing to the sterile diet.
Leave the caps loose to allow oxygen to enter the tube. The transferred eggs will be visible on the surface of the diet. 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 full 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 of 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 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.
Each of the four species in the gnotobiotic Drosophila melanogaster presents different morphologies. Acetobacter colonies are a clear light brown, and come in varying sizes. A.tropicalis is opaque, whereas A.pomorum is transparent.
L.Brevis colonies are small and white, and L.plantarum colonies are large and yellow. This graph displays the colony-forming units, or CFUs per fly, of four bacterial strains isolated from axenic and gnotobiotic Drosophila. Lack of colonies in the axenic homogenates confirms D.melanogaster sterility.
Once mastered, microbe-free Drosophila can be prepared in under an hour, if performed properly. This procedure can be performed with bacterial strains other than those described in this video, in order to explore the effects of myriad bacteria on Drosophila melanogaster. Instead of starting with microorganism-free Drosophila, a bacterial cocktail can be inoculated to conventional Drosophila stocks to ensure that flies in each vial have access to a shared set of microbes.
