This article describes a method for infecting Drosophila melanogaster with bacterial suspensions using a nanoinjector. The process involves precise injection techniques to minimize damage and ensure effective bacterial delivery into the fly's hemocoel.
To infect flies, begin with a narrow-diameter glass needle filled with mineral oil.
Secure the needle onto the nanoinjector plunger with an O-ring and a spacer for precise and controlled injection, ensuring minimal damage to the flies.
Release the mineral oil, leaving a small amount in the needle as a barrier between the bacterial solution and the injector. Load the needle with the bacterial suspension.
Position an anesthetized Drosophila melanogaster ventrolateral side-up.
Inject the needle into the fly's anterior abdomen, piercing through the cuticle and epithelial layer. Deliver the bacterial suspension into the hemocoel — a body cavity containing hemolymph — a nutrient-rich circulatory fluid.
Within the hemocoel, immune cells called hemocytes, circulate alongside the fat body, a specialized tissue equipped with pattern recognition receptors.
Allow the injected flies to recover in a vial containing medium. Injected bacteria utilize the nutrients from hemolymph and multiply, increasing the bacterial load, and establish a systemic infection.
Fat body cell receptors interact with bacterial antigens, triggering an immune response to produce antimicrobial peptides, ultimately eliminating some bacteria.
Circulating hemocytes perform phagocytic activity, engulfing and degrading a few bacteria. However, some bacteria evade the host's immune response and survive within the flies, establishing a bacterial infection.
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.
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