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

- Fast an adult fish for 24 hours before injection to empty the gastrointestinal tract and create additional space in the abdomen. On the day of the procedure, anesthetize the fish with tricaine and place it into a slot in a wet sponge with the ventral side facing up.

Next, pipette the desired amount of injection solution onto a piece of laboratory film and pull it into an insulin needle. Position the needle between the pelvic fins at a 45 degree angle from the anterior-posterior body axis and gently push it in at the midline. Advance the needle approximately 1 to 2 millimeters into the abdominal cavity, the space between the abdominal wall and internal organs, and slowly inject the solution of interest. Wait for five seconds before pulling out the needle to avoid any spillage. Now, carefully remove the needle.

After injection, quickly transfer the fish into a recovery tank containing fresh tank water. If the fish doesn't begin to swim immediately, swirl the water near the gills to help it recover from anesthesia. In the example protocol, we will inject a mycobacterium marinum solution into RAG1 mutant fish to study infection.

- First, pipe out of 5 microliter droplet of the diluted bacterial solution onto a piece of paraffin film. Then, pull the droplet into a 30 gauge insulin needle. Use a five to eight-month-old fish for this experiment, with one being a wild-type fish and the other being a RAG mutant fish.

Position these fish ventral side up in the slits of a piece of moist foamed plastic. Inject the insulin needle between the pelvic fins at a 45 degree angle. Keep the needle opening upwards to ensure that the entire opening is inside the abdominal cavity. Then, slowly inject the bacterial solution. After this, carefully remove the needle and immediately transfer the fish to a recovery tank filled with fresh tank water.

Take samples from the bacterial aliquot in use every 15 minutes on 7H10 plates. Incubate these samples at 29 degrees Celsius for five days to verify the infection dose. Check the well-being of the fish regularly, making sure to euthanize any fish with infection symptoms by incubating them in water with more than 0.02% of 3-aminobenzoic acid ethyl ester.