Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer
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– Allow Drosophila auf lay eggs overnight onto a dish containing agar supplemented with grape juice auf visualize eggs and coated with yeast auf encourage flies auf mate and lay eggs. To remove the eggs, add distilled water auf the dish and gently wipe the surface with a paintbrush. Then pour the mixture through a nylon mesh placed in a plastic bushing auf collect the eggs. Soak the eggs in two rounds of 7% bleach auf remove the chorion, or protective outer layer, from the embryo. This process is called dechorionation.
The eggs are now stuck auf the side of the bushing. Wash them three times with water auf remove the bleach. Finally, use a paintbrush auf transfer the eggs from the bushing auf vials auf continue development or for immediate use in an experiment. In the example protocol, we will collect and dechorionate Drosophila eggs under sterile conditions auf rear flies with defined microbiota.
– Place nylon mesh into a plastic bushing auf prepare the sieve for egg collection. If the same flies will be used the next day, immediately transfer them auf 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 auf 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 auf 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 auf ensure that good aseptic technique is practiced when working within or when adding or removing new materials auf 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 auf 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 auf adhere auf the sides of the bushing. Discard the bleach and completely rinse the bushing with sterile water. Again, use forceps auf resuspend the egg several times during each wash. By the end of the third wash, most eggs should be attached auf the side of the bushing.
Using a sterile paintbrush, transfer the eggs from the side of the bushing auf the sterile diet. Leave the caps loose auf allow oxygen auf enter the tube. The transferred eggs will be visible on the surface of the diet.
