This protocol addresses the difficulties and challenges with microinjecting larva by describing a precise method that will allow the use of Drosophila melanogaster larva as a model for various immune and molecular studies. This is a simple injection technique that presents an efficient, cost-effective and rapid method to repeatedly deliver a variety of substances in a uniform manner, which allows for consistent and reliable results. Begin by selecting third instar stage larvae and washing them with Ringer's solution in a small Petri dish.
Place a filter paper in a Petri dish and moisten it with 5 ml of Ringer's solution. Then place the larvae on the filter paper. Prepare capillaries using 3.5"glass capillary tubes in a micropipette puller.
Open the glass capillary with the help of straight serrated medium point forceps. Fill the open capillary with mineral oil using a plastic disposable syringe. Then, eject the oil out of the capillary tube with a nanoliter injector.
Fill the capillary with the desired bacterial preparation for injection. Transfer the anesthetized larvae to a filter paper moistened with Ringer's solution. Using forceps, apply firm pressure on the dorsal side of the posterior end of the larvae, where the tracheal spiracles are apparent.
Then, insert the needle horizontally, towards the posterior end of the larvae, and inject the bacterial stocks. Remove forceps to avoid spillage of hemolymph or intestine. Then, withdraw the needle previously inserted into larvae.
Infect 20 larvae for each experimental condition. Using a paintbrush, gently transfer the injected larvae to a separate moist filter paper. Add Ringer's solution as necessary to prevent desiccation.
Incubate the Petri dish in an incubator at 25 C.Drosophila melanogaster larvae infected with Photorhabdus asymbiotica exhibited a 57%survival rate for 24 hours following injection, while larva injected with Escherichia coli showed an 85%survival rate at the same time point. The importance of this method is that the needle is held approximately parallel to the larva. The pressure applied to hold the larva is very little, which reduces the possibility of hemolymph leakage, fatal injury, or inadequate delivery of the desired substance.
This technique can be perfected with practice.
