Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer
Transcript
– Allow Drosophila to lay eggs overnight onto a dish containing agar supplemented with grape juice to visualize eggs and coated with yeast to encourage flies to mate and lay eggs. To remove the eggs, add distilled water to the dish and gently wipe the surface with a paintbrush. Then pour the mixture through a nylon mesh placed in a plastic bushing to collect the eggs. Soak the eggs in two rounds of 7% bleach to remove the chorion, or protective outer layer, from the embryo. This process is called dechorionation.
The eggs are now stuck to the side of the bushing. Wash them three times with water to remove the bleach. Finally, use a paintbrush to transfer the eggs from the bushing to vials to continue development or for immediate use in an experiment. In the example protocol, we will collect and dechorionate Drosophila eggs under sterile conditions to rear flies with defined microbiota.
– 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 paintbrush, and take caution not to break up the agar. Rinse the agar plate with distilled water and gently brush the eggs from the agar’s 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 resuspend 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 resuspend the egg 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 paintbrush, 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.
