Establishing Bacterial Infection in Drosophila Larvae

Samenvatting

Source: Hiroyasu, A., D et. al., Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis. J. Vis. Exp. (2018)

This video demonstrates the procedure for establishing a bacterial infection in Drosophila larvae by placing them into a bacterial droplet and pricking them with a tungsten needle. The larvae are then incubated on a grooved, nutrient-rich agar plate to study host-pathogen interactions.

Protocol

1. In vivo infection

1. Warm the Drosophila fruit juice agar plate at room temperature for 15 min.
   1. Add 30 g of agar to 700 mL of water and autoclave it for 40 min.
   2. Dissolve 0.5 g of methyl paraben in 10 mL of absolute ethanol.
   3. Add the methyl paraben solution to 300 mL of fruit juice concentrate.
   4. Quickly mix the juice concentrate into the autoclaved agar solution and dispense 5 mL into 10 × 35 mm Petri dishes.
   5. After the plates have cooled for 15 min, store them at 4 °C.
2. Prepare 3rd instar larvae.
3. Place the yeast paste on an agar plate. Make a fine cut in the agar plate where larvae can migrate to avoid drying (Figure 1A). Assemble a 0.001 mm pointed tungsten needle with holding forceps using paraffin film (Figure 1B, C).
4. Pipette 50 µL of high-titer mCherry expressing-Coxiella burnetii (5.95×10⁹ GE/mL) onto the paraffin film under the stereo microscope and place the larvae into the pool of bacteria.
5. Place the larvae in the pool of bacteria, prick them with a tungsten needle (Figure 3D), and transfer them onto an agar plate (Figure 1E).
6. Transfer the remaining pathogen medium onto the agar plate and seal the plate with paraffin film.
7. Keep the larvae on the plate in moist air until the desired time post-infection (Figure 1F). In this experiment, C. burnetii-infected larvae are on the plate for 24 h.

Resultaten

Figure 1: In vivo infection. A) Drosophila fruit juice agar plates and yeast paste...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Stereo microscope	Amscope	SM-1BSZ-L6W
Forceps	VWR	82027-402
Coxiella burnetii - mCherry	Dr. Heinzen, R.
Drosophila fruit juice plates	Cold Spring Harbor Protocols		http://cshprotocols.cshlp.org/content/2007/9/pdb.rec11113.full
Agar	Fisher Bioreagents	BP1423-500
Methyl paraben	Amresco	0572-500G
Absolute ethanol	Fisher Bioreagents	BP2818-500
Welch's 100% Grape juice frozen concentrate, 340 mL	Amazon	B0025UJVGM
Petri dishes, 10 x 35 mm	Fisher Scientific	08-757-100A
Yeast, Bakers Dried Active	MP Biomedicals	210140001	Add 2 parts of water to 1 part of yeast (v/v)
Tungsten needle	Fine Science Tools	10130-20
Holding forceps	VWR	HS8313
Listeria monocytogenes	ATCC	strain: 10403S	Listeria monocytogenes strain 10403S (Bishop and Hinrichs, 1987) was grown in Difco Brain-heart infusion (BHI) broth (BD Biosciences) containing 50 µg/ml streptomycin at 30 °C.