The primary advantage of this assay is the simplicity and the accuracy because it does not require an embryo rinse to transfer to the food vial and that avoids losses from technical errors. This protocol does not require expert technical skills. However, the proper timing and careful water transfer are important for its accuracy and reproducibility.
Demonstrating this procedure will be Antonio Rockwell, a recently graduated PhD student from my laboratory. Before beginning the procedure, pour grape agar into a 35-millimeter Petri dish to half full and allow the agar to solidify for one hour. When the agar has solidified, use a small plastic knife to gently scratch the surface of the agar around the outside of the plate, leaving the middle of the plate free of scratches.
Then place a small amount of freshly prepared yeast paste into the center of the dish and place the dish into an embryo collection mini cage. For embryo collection, first place two virgin Drosophila females and one young male inside the collection cage for 24 hours. The next day, inspect the bottom of the agar plate to look for embryos.
If embryos have been laid, transfer the agar plate into a humid chamber and cover the plate with the lid to avoid dehydration. If several days of laying are to be scored, place a new agar plate into the collection cage. Use the microscope to determine the number of hatched embryos and L1 larvae, and return the embryos to the humid chamber at room temperature until all of the embryos have hatched and developed into L1 larvae.
After 48 hours, count the number of hatched embryos and L1 larvae and use a spatula to carefully remove the disc of grape agar from the Petri dish, placing the disk L1 side down on the food in a food vial large enough to accommodate the disc. Then carefully inspect the Petri dish for any remaining larvae. Monitor the food vial daily at roughly the same time each day to confirm that the L2 and L3 larvae can be observed making their way to the food.
Record the number of pupae and adult Drosophila, continuing to count until no more adults are observed and avoiding counting the subsequent generation. Then perform a chi-square analysis. The null hypothesis assumes a 100%viability, such that the number of adults will be equal to the number of hatched embryos and L1 originally recorded and transferred to the food vials.
When the agar is placed on the side of the vial, many of the embryos and larvae do not mature to adults, likely due to the grape agar disc dehydration. When the grape agar is placed face-down inside the vial in direct contact with the food surface, less than 6%of the progeny is typically lost, as the agar remains hydrated while in contact with the moisture of the instant food inside the vial. In this representative experiment comparing wild-type and mutant progeny development, approximately 91%of the mutant progeny matured to adulthood using this method compared to 94%of the progeny from wild-type flies.
Remember to transfer the agar disc embryo-side down into the food vial and then, to check the empty Petri dish for any remaining larvae that did not get transferred. Another crucial step is transferring the agar disc to a food vial no later than 48 hours after removing the grape agar plate from the embryo collection mini cages to prevent dehydration.
