Infecting Flies with Bacterial Suspension Using a Nanoinjector

1. Collect and Prepare Flies

1. Rear Drosophila 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 before 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 before 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 Sodium chloride, NaCl, 2.7 mM potassium chloride, KCl, 10 mM sodium phosphate, Na₂PO₄, 1.8 mM potassium dihydrogen phosphate, 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. Using a Nanoinjector
   1. Prepare the glass needles for infection.
      1. Prepare a glass needle by pulling a borosilicate glass capillary using a micropipette puller.
      2. Using forceps, break off the tip of the needle to create an opening of approximately 50 µm diameter to allow the ejection of liquid.
   2. Assemble the injector.
      1. Place the sealing O-ring and then the white spacer (large dimple facing outwards) onto the metal plunger.
      2. Fill the glass needle with mineral oil using a syringe with a 30 G needle.
      3. Put the filled glass needle through the collet and then place the larger O-ring around its base, about 1mm from the blunt end of the needle.
      4. Slide the needle onto the metal plunger and gently screw on the collet until secure.
      5. Eject most of the mineral oil from the injector, but make sure that there is still 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 or bacterial suspension, or between the two liquids.
      6. Set the injector to the desired volume for injection (between 9 nl and 50 nl).
   3. Generate wounding controls.
      1. Fill the injector needle with sterile PBS by carefully inserting the tip of the capillary needle in a tube of the media and pressing the "fill" button on the injector.
         NOTE: Sterile bacterial growth media can also be used as wounding control if the experiment requires injection of bacteria suspended in their growth medium. However, because bacterial growth media contains components that may stimulate the immune system or have other effects on the host, an inert carrier such as PBS is preferable.
      2. Anaesthetize the desired number of flies under a light flow of carbon dioxide (CO₂).
      3. Inject the flies in the anterior abdomen on the ventrolateral surface with the sterile PBS.        
         NOTE: It is also possible to inject flies at other sites, such as the sternopleural plate of the thorax, but it is important to keep the injection site consistent within each experiment.
      4. Place injected flies into fresh vials with a new medium, laying the vials on their side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food.        
         NOTE: It is recommended to inject the PBS control flies before injecting bacteria into experimental flies so the same needle can be used for both treatments. It will not always be possible to use the same needle for an entire experiment. In that case, it may be desirable to record which needle was used with which flies and to include needle identity as an experimental factor in statistical analysis.
   4. Inject the bacterial pathogen.
      1. Eject the remaining sterile media from the injector and refill the same needle with the bacterial suspension.
      2. Repeat the above procedure (3.1.3), now injecting the flies with the bacterial suspension prepared in procedure 2.2.