Establishing Bacterial Infection in Drosophila Larvae

Take a droplet of a bacterial pathogen on a paraffin film.

Place a Drosophila larva in the bacterial droplet.

Under a stereomicroscope, use a tungsten needle to prick the larva and puncture its cuticle, the protective outer layer, and the underlying epidermis.

This allows bacterial entry into the larval hemolymph, the circulatory fluid.

Transfer the infected larvae onto a grooved, nutrient-rich agar plate containing yeast paste as a food source.

The grooves facilitate larval migration and prevent dehydration.

Add the remaining bacterial suspension to the plate and seal it with paraffin film.

Incubate the plate in a moist chamber to maintain larval viability.

Inside the larval hemolymph, immune cells called hemocytes detect the bacteria and initiate phagocytosis.

While some bacteria are eliminated after phagosome–lysosome fusion, others survive by delaying fusion or resisting intracellular stress and replicate within hemocytes, leading to a sustained intracellular infection.

The infected Drosophila larvae are now ready for host-pathogen interaction studies.

Place yeast paste on a grape juice agar plate. Make a fine cut in the agar where the larvae can migrate to avoid drying.

Assemble a 0.001 millimeter pointed Tungsten needle with holding forceps using paraffin film. Pipette 50 microliters of high-titer, mCherry-expressing C. Burnetii onto the paraffin film under the stereo microscope, and place the larvae into the pool of bacteria. Prick the larvae with a Tungsten needle.

Transfer the larvae onto an agar plate. Transfer the remaining pathogen medium onto the agar plate, and seal the plate with paraffin film. Keep the larvae on the plate in moist air until the desired time post-infection. Leave the C. Burnetii infected larvae on the plate for 24 hours.