Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer

– Allow Drosophila 到 lay eggs overnight onto a dish containing agar supplemented with grape juice 到 visualize eggs and coated with yeast 到 encourage flies 到 mate and lay eggs. To remove the eggs, add distilled water 到 the dish and gently wipe the surface with a paintbrush. Then pour the mixture through a nylon mesh placed in a plastic bushing 到 collect the eggs. Soak the eggs in two rounds of 7% bleach 到 remove the chorion, or protective outer layer, from the embryo. This process is called dechorionation.

The eggs are now stuck 到 the side of the bushing. Wash them three times with water 到 remove the bleach. Finally, use a paintbrush 到 transfer the eggs from the bushing 到 vials 到 continue development or for immediate use in an experiment. In the example protocol, we will collect and dechorionate Drosophila eggs under sterile conditions 到 rear flies with defined microbiota.

– Place nylon mesh into a plastic bushing 到 prepare the sieve for egg collection. If the same flies will be used the next day, immediately transfer them 到 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 到 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 到 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 到 ensure that good aseptic technique is practiced when working within or when adding or removing new materials 到 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 到 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 到 adhere 到 the sides of the bushing. Discard the bleach and completely rinse the bushing with sterile water. Again, use forceps 到 resuspend the egg several times during each wash. By the end of the third wash, most eggs should be attached 到 the side of the bushing.

Using a sterile paintbrush, transfer the eggs from the side of the bushing 到 the sterile diet. Leave the caps loose 到 allow oxygen 到 enter the tube. The transferred eggs will be visible on the surface of the diet.