Infecting Flies with Bacterial Suspension Using a Nanoinjector

Prepare a glass needle by pulling a borosilicate glass capillary, and use forceps to break off the tip of the needle to create a 50-micrometer diameter opening. Next, place the sealing O-ring and the white spacer, with the large dimple facing outward, onto the metal plunger of the injector. Fill the glass needle with mineral oil using a syringe with a 30-gauge needle.

Then, place the filled glass needle through the collet and place the larger O-ring around its base, 1 millimeter from the blunt end of the needle. Slide the needle onto the metal plunger and gently screw on the collet until it is secure.

Dispel the mineral oil from the injector leaving a small volume of oil in the needle to act as a barrier between the injector and the bacterial suspension. Make sure that there are no air bubbles within the mineral oil, bacterial suspension, or between the two liquids. Then, set the injector to the desired volume for injection. Carefully insert the tip of the capillary needle in a tube of sterile PBS, and press the fill button on the injector to fill the injector needle.

To generate wounding controls, inject anesthetized flies with sterile PBS in the anterior abdomen on the ventrolateral surface. Place the injected flies into fresh vials with new media, and lay the vials on their side to prevent the flies from becoming stuck to the food before they recover from their anesthesia. Next, eject the remaining sterile media from the injector, and refill the same needle with the bacterial suspension. Repeat the procedure and inject the flies with the bacterial suspension.