Intraperitoneal Injection: A Method of Solution Delivery into the Abdominal Cavity of an Adult Zebrafish

1.  Experimental M. marinum Infection with Intraperitoneal Injection

1. Pipette a 5 µL droplet of the diluted bacterial solution onto a piece of parafilm film and pull the droplet into a 30 G insulin needle.
2. Use 5–8 month-old wild-type fish and rag1−/−hu1999 mutant fish for the experiment. Anesthetize adult zebrafish in the tank water with 0.02% 3-aminobenzoic acid ethyl ester (pH 7.0). Position the fish ventral side up into a slit on a moist foamed plastic.
   NOTE: Rag-/- mutant fish are not able to undergo somatic recombination and produce functional T and B cells.
3. Inject the needle between the pelvic fins at an approximately 45° angle. Keep the needle opening upwards to observe that the entire opening is inside the abdominal cavity. Slowly inject the bacterial suspension and carefully remove the needle.
   NOTE: In case the red tracer is leaking out of the fish upon injection, exclude the fish from the experiment.
4. Immediately after injection, transfer the fish into a recovery tank with fresh tank water.
5. Take samples of the bacterial suspension on 7H10 plates every 15 min from the bacterial aliquot in use and incubate the bacteria at 29 °C for 5 days and verify the infection dose by counting the colonies on the plates.
6. Check the well-being of the fish regularly and euthanize any fish with symptoms of the infection with over 0.02% concentration of 3-aminobenzoic acid ethyl ester (pH 7.0).
   NOTE: Approximately, 7% of the adult zebrafish infected with 34 ± 15 CFU and 30% of zebrafish infected with 2029 ± 709 CFU will have had symptoms by 8 weeks. The symptoms may include abnormal swimming, lack of response to touch, gasping, edema or observable wasting.
7. Maintain the zebrafish according to the common standards.