1.&#160; Experimental M. marinum Infection with Intraperitoneal Injection
<ol>
	<li>Pipette a 5 &#181;L droplet of the diluted bacterial solution onto a piece of parafilm film and pull the droplet into a 30 G insulin needle.</li>
	<li>Use 5&#8211;8 month-old wild-type fish and rag1&#8722;/&#8722;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.</li>
	<li>Inject the needle between the pelvic fins at an approximately 45&#176; 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.</li>
	<li>Immediately after injection, transfer the fish into a recovery tank with fresh tank water.</li>
	<li>Take samples of the bacterial suspension on 7H10 plates every 15 min from the bacterial aliquot in use and incubate the bacteria at 29 &#176;C for 5 days and verify the infection dose by counting the colonies on the plates.</li>
	<li>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 &#177; 15 CFU and 30% of zebrafish infected with 2029 &#177; 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.</li>
	<li>Maintain the zebrafish according to the common standards.</li>
</ol>

<table><tbody><tr><td>Mycobacterium marinum&amp;nbsp;</td><td>American Type Culture Collection&amp;nbsp;</td><td>ATCC 927</td><td></td></tr><tr><td>Phenol red</td><td>Sigma-Aldrich</td><td>P3532</td><td></td></tr><tr><td>Phosphate buffered saline tablets (PBS)</td><td>Sigma-Aldrich</td><td>P4417-50TAB</td><td></td></tr><tr><td>Middlebrock 7H10 agar</td><td>BD, Thermo Fisher Scientific</td><td>11799042</td><td></td></tr><tr><td>3-aminobenzoic acid ethyl ester (pH 7.0)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Omnican 100 30 G insulin needle</td><td>Braun</td><td>9151133</td><td></td></tr><tr><td>1 mL syringe</td><td>Henke Sass Wolf</td><td>4010.200V0</td><td></td></tr></tbody></table>

intraperitoneal injection: a method of solution delivery into the abdominal cavity of an adult zebrafish

Source: Luukinen, H., et al. <a href="https://www.jove.com/v/58299/" target="_blank">Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish</a>. J. Vis. Exp. (2018).
This video describes a technique of intraperitoneal injection, a method for delivering solutions into the abdominal cavity of Adult Zebrafish.

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

Source: Luukinen, H., et al. Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish.  J. Vis. Exp. (2018).
This video describes a ...

- 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&#39;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.

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Danio rerio (zebrafish)

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8.3K Views. - Fast an adult fish for 24 hours before injection to empty the gastrointestinal tract and create additi...

Abstract

Protocol

<table><tbody><tr><td>Mycobacterium marinum&nbsp;</td><td>American Type Culture Collection&nbsp;</td><td>ATCC 927</td><td></td></tr><tr><td>Phenol red</td><td>Sigma-Aldrich</td><td>P3532</td><td></td></tr><tr><td>Phosphate buffered saline tablets (PBS)</td><td>Sigma-Aldrich</td><td>P4417-50TAB</td><td></td></tr><tr><td>Middlebrock 7H10 agar</td><td>BD, Thermo Fisher Scientific</td><td>11799042</td><td></td></tr><tr><td>3-aminobenzoic acid ethyl ester (pH 7.0)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Omnican 100 30 G insulin needle</td><td>Braun</td><td>9151133</td><td></td></tr><tr><td>1 mL syringe</td><td>Henke Sass Wolf</td><td>4010.200V0</td><td></td></tr></tbody></table>

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

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Name	Company	Catalog Number
Mycobacterium marinum	American Type Culture Collection	ATCC 927
Phenol red	Sigma-Aldrich	P3532
Phosphate buffered saline tablets (PBS)	Sigma-Aldrich	P4417-50TAB
Middlebrock 7H10 agar	BD, Thermo Fisher Scientific	11799042
3-aminobenzoic acid ethyl ester (pH 7.0)	Sigma-Aldrich	A5040
Omnican 100 30 G insulin needle	Braun	9151133
1 mL syringe	Henke Sass Wolf	4010.200V0