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