$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
This protocol is an excerpt from Koyle et al., Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions, J. Vis. Exp. (2016).
1. Prepare Egg-laying Cages
- Make grape-juice agar plates by microwaving 100 ml water, 10 g brewer's yeast, 10 g glucose, and 1 g of agar. Bring to a boil 3 times in step 1.1 and add 10 g of frozen grape juice concentrate to increase visibility of eggs on the agar plate.
- When agar has cooled to 55 °C, pour 20 ml into 100 mm Petri dishes and allow to solidify.
- Cover the surface of the agar plates with a yeast paste by mixing 1 g brewer's yeast with 15 g water. Pour yeast paste onto the agar plate making sure the surface is covered, then pour off the excess, leaving a thin yeast residue behind. If multiple plates are used, the paste can be sequentially poured to multiple plates.
- Transfer agar plates into the bottom of a cage.
NOTE: A 32 oz deli container is a good substitute for acrylic fly cages.
- Make lids for 32 oz deli containers by cutting a hole in the top and gluing a breathable mesh over the hole with non-toxic glue. If the glue is water soluble prevent water exposure to the lid.
- Transfer 200-300 flies into the container and cover with lid. Cover the mesh-protected hole with an empty Petri dish lid to prevent evaporation from the agar surface and add a moist tissue paper inside the lid if desired. Incubate flies at 25 °C overnight for 16-20 hr.
2. Collect Eggs
- Prepare a sieve for egg collection by placing nylon mesh into a plastic bushing.
- To retrieve the plate with eggs on it, remove flies from the cage by transferring to an empty container. If same flies will be used the next day, transfer immediately to a new cage containing a freshly yeasted grape-juice agar plate. Flies can be used for 3-5 sequential days
- Remove dead flies from the agar with a clean paintbrush, being careful to not break up the agar.
- Collect eggs by rinsing the agar plate with distilled water, gently brushing eggs from the agar surface, and pouring the slurry over the mesh. Repeat 3-4 times until all or most of the eggs have been removed from the agar plate.
3. Dechorionate Eggs and Transfer to Sterile Diet
- Prepare the biosafety cabinet by spraying the inside (including sides) with 70% ethanol. Wipe the bottom with a lab tissue, and sterilize the hood with UV light for ~15 min. Sterilize all non-biological supplies (specimen cups, paintbrush, forceps, waste container, 400 ml sterilized water, and 100 ml of 0.6% sodium hypochlorite) by spraying with ethanol and immediately placing in the biosafety cabinet. Sterilize with UV light for 15 min.
- Start the first of 2 sodium hypochlorite washes by placing the bushing with the eggs into a 120 ml specimen cup or other sterile container. Slowly pour ~90 ml of 0.6% sodium hypochlorite solution into the bushing until just below the rim.
- Rinse eggs for 2.5 min. Periodically re-suspend the eggs by using forceps to move the bushing up and down in the hypochlorite solution.
- Transfer the bushing directly into a second specimen cup, pre-filled with 90 ml bleach, inside the biosafety cabinet.
- Repeat step 3.3 inside the biosafety cabinet. At the end of the second bleach treatment, the eggs should begin to adhere to the sides of the bushing.
- Carry out steps 3.7-3.8 in the biosafety cabinet.
- Discard the bleach and wash the bushing with sterile water 3 times. Re-suspend the eggs several times during each washing by moving the bushing with forceps. By the end of the third washing most eggs should be attached to the side of the bushing.
- Using a paintbrush sterilized in ethanol, transfer eggs from the side of the bushing to the sterile diet. Transfer eggs individually or in small batches. Aim for 30-50 eggs per vial. Leave the caps loose to allow oxygen to enter the tube.