Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer
Transcripción
– Allow Drosophila a lay eggs overnight onto a dish containing agar supplemented with grape juice a visualize eggs and coated with yeast a encourage flies a mate and lay eggs. To remove the eggs, add distilled water a the dish and gently wipe the surface with a paintbrush. Then pour the mixture through a nylon mesh placed de a plastic bushing a collect the eggs. Soak the eggs de two rounds of 7% bleach a remove the chorion, or protective outer layer, from the embryo. This process is called dechorionation.
The eggs are now stuck a the side of the bushing. Wash them three times with water a remove the bleach. Finally, use a paintbrush a transfer the eggs from the bushing a vials a continue development or for immediate use de an experiment. In the example protocol, we will collect and dechorionate Drosophila eggs under sterile conditions a rear flies with defined microbiota.
– Place nylon mesh into a plastic bushing a prepare the sieve for egg collection. If the same flies will be used the next day, immediately transfer them a a new cage containing freshly yeasted grape juice agar plates. The grape juice plate de the previous cage contains the fruit fly eggs. Remove dead flies with a clean paintbrush, and take caution not a 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 a 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 de the biosafety cabinet, then sterilize them with UV light.
– It is critical a ensure that good aseptic technique is practiced when working within or when adding or removing new materials a 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 de the solution a 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 a adhere a the sides of the bushing. Discard the bleach and completely rinse the bushing with sterile water. Again, use forceps a resuspend the egg several times during each wash. By the end of the third wash, most eggs should be attached a the side of the bushing.
Using a sterile paintbrush, transfer the eggs from the side of the bushing a the sterile diet. Leave the caps loose a allow oxygen a enter the tube. The transferred eggs will be visible on the surface of the diet.
