A Septic Pinprick Technique to Introduce Systemic Bacterial Infection in Flies

1. Collect and Prepare Flies

1. Rear D. melanogaster under the desired experimental conditions. Take care not to overcrowd the flies during rearing and make sure that larval densities are consistent across treatments since environmental conditions during the larval stage can profoundly affect immune defense phenotypes during the adult stage.
2. Collect experimental flies 0 – 3 days after eclosion from the pupal case and transfer them onto fresh medium.
3. House the collected flies at a desired temperature (temperatures between 22 °C and 28 °C are generally suitable) until they are aged 5 – 7 days post-eclosion.
   NOTE: This allows sufficient time for the flies to complete metamorphosis and become mature adults, but is well before senescence begins.
4. Sort the desired number of flies into a separate vial prior to infection, again taking care to avoid overcrowding.
   NOTE: Only males are infected in this demonstration but is equally possible to infect females using the procedure described.

2. Culture and Prepare Bacteria

1. Prepare a master plate of the chosen bacteria at least 2 days prior to infection. Streak the bacteria from a 15% glycerol stock stored indefinitely at -80 °C. Store the master plate at 4 °C for up to 2 weeks. Always streak the master plates directly from the frozen glycerol stock. Avoid serial passage of bacteria from plate to plate, as repeated passage in culture can cause bacteria to evolve attenuated virulence.
   NOTE: This example makes use of Providencia rettgeri and Escherichia coli.
2. Use the following procedure to prepare a bacterial suspension for injection.
   1. Grow a 2 ml culture of bacteria by inoculating a sterile medium with a single colony isolated from the master plate. Grow the bacteria to the stationary phase (e.g. O/N growth at 37 °C).
      NOTE: Both P. rettgeri and E. coli grow well in Luria Broth at temperatures from 20 – 37 °C with gentle shaking.
   2. Once the culture has reached the stationary phase, gently pellet cells from approx. 600 µl of the culture in a tabletop centrifuge (3 min at 5,000 x g), discard the supernatant and resuspend the bacteria in approx. 1,000 µl of sterile phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂PO₄, 1.8 mM KH₂PO₄, pH = 7.4).
   3. Dilute the resuspended cells in sterile PBS to achieve an optical density (OD) appropriate for the bacterium being used, measured with a spectrophotometer as the absorbance at 600 nm. Use this suspension to infect the flies.
      NOTE: A600 = 0.1 or 1.0 corresponds respectively to approximately 10⁸ and 10⁹ Providencia rettgeri or E. coli per ml. Because both live and dead bacteria contribute to optical density, it is important to harvest cultures early in the stationary phase before bacterial corpses accumulate and distort the relationship between optical density and the number of viable bacteria introduced during infection.

3. Infect the Flies

NOTE: As Drosophila immunity is influenced by circadian rhythm, it is important to perform infections at a similar time of day across experimental replicates.

1. With Septic Pinprick
   1. Prepare the needle for pricking.
      1. Melt the end of a 200 µl micropipette tip and insert a 0.15 mm insect minutien pin into the molten plastic. Allow the plastic to solidify such that the pin is held in place with approximately 0.5 cm of the pin extending from the plastic.
   2. Generate wounding controls.
      1. Anesthetize the desired number of flies under a light flow of CO₂.
      2. Prick the flies in the sternopleural plate of the thorax with the needle, avoiding the attachment sites of the wings and the legs. If necessary, gently remove flies from the minutien pin using soft forceps.
         NOTE: It is also possible to prick the flies at other sites, such as the anterior abdomen on the ventrolateral surface, but it is important to keep the injection site consistent within each experiment. Pricking through the cuticle of the abdomen tends to be more difficult than pricking the thorax and is therefore less common.
      3. Place the pricked flies into a fresh vial with a new fly medium, laying the vial on its side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food.
   3. Introduce the bacterial infection.
      1. Anesthetize the desired number of flies under a light flow of CO₂.
      2. Prick each fly in the same location as the media controls, dipping the tip of the pin into the bacterial suspension prepared in procedure 2.2 before pricking each fly.
      3. Place the pricked flies into a fresh vial with a new fly medium, laying the vial on its side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food.