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everal methods of anesthesia have been historically used with fishes. The most common means of anesthesia involves use of a commercial anesthetic called MS-222 (3-Aminobenzoic Acid Ethyl Ester Methanesulfonate salt, also called ethyl m-aminoboenzoate; C₉H₁₁NO₂-CH₄SO₃; Sigma Cat # A-5040 or equivalent). A 0.4% stock solution is typically made in tris-buffered water as follows:

MS-222 stock solution (0.4%, 100 ml):

400 mg tricaine
97.9 ml autoclaved/distilled water
2.1 ml 1 M Tris -Cl (pH 9)

Adjust pH to 7.0. Store this solution in a dark container. Some individuals freeze this stock solution, while others keep it at room temperature. Regardless of storage parameters, tricaine can get old and lose potency.

Please note the use of Tris as a buffer. This is recommended, since drammatic changes of pH can occur when making MS-222 solutions.

Changes in pH when making unbuffered MS-222 solutions

Changes in pH when making Tris-buffered MS-222 solutions

MS-222 working solution (0.02%, 100 ml):
To use tricaine as an anesthetic combine the following in a Lee specimen container:

5 ml MS-222 stock solution
95 ml clean tank water (preferably from the same tank that the fish came from)

Anesthesia of fish:

Anesthetize a fish by placing it into a dedicated anesthetic container with 100 ml of 0.02% anesthetic (MS-222; in water). The swimming movements of the fish will slow down gradually (approximately 30 seconds, depending on the size of the animal). It will lose its ability to stay upright in the water and keel over on its side or totally lie on the bottom upside down. The mouth and gill arches will still be moving, albeit very slowly. It will take ~2 minutes for the fish to become fully anesthetized, at which point it should be removed with a wet gloved hand and placed on the wet cotton under the microscope. The fish is extremely vulnerable to injury, particularly around the ocular area, so take care not to scrape the fish against the sides of the container. The fish will remain anesthetized for approximately 2 minutes.

Recovery:

The fish should be placed back in its original tank water, and carefully monitored for signs of recovery. The fish will initially show slow movement of the mouth and gill area. If it does not, the fish has been too deeply anesthetized. If this is the case, physically move water over the opercular region. The fish should eventually begin more rapid opercular movements and right itself in the water.

Contributed by Steven Kazianis, David Trono, Leona C. Hazlewood and Ronald Walter.

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