SOP-4 Tricaine Anesthetic Preparation

August 19, 2016 (Version 1)

S.O. P- 4 Synopsis
Tricaine-S Methanesulfonate Anesthetic Solution Preparation

Name of Biola University Zebrafish Lab

Institution/sponsor:

Primary Objective: The Primary objective of this SOP is to prepare Tricaine-S solution for
Anesthesia and Tranquilization of study subjects for transport, surgical
procedures, weighing, and other proper handling required during research

Secondary Objective: To prepare anesthetic solution comprised of Tricaine-S solution to

concentrations required for surgery and physiological studies of zebrafish.

Active Spring water or conditioned water 48 ml, ethyl-amino benzoate (Tricaine-S

Chemicals/Ingredients: Methanesulfonate 0.2 g, NaOH 0.2 g

4.0 Introduction
Tricaine-S is an isomer of benzocaine having the formula C9H11O2N + CH3SO3H.

Its primary function in causing anesthetic effects in fish is through the distruption of sodium penetration
into the nerve cell. Nerve cells dependability on Na+ for excitability is highly relient on the concentration
of Na+ crossing the cell membrane. Tricaine-S moves easily through the lipid membrane binding the
sodium Ion channels suppressing nerve function. This results in a loss of “communication” between the
nerve cells on stimulus from pain, temperature, and loss of equilibrium (as studied in mammals). These
pharmacokinetics have not been extensively studied in fish and thus the predicted effects of Tricaine-S
have been studied, recorded and utilized as the parameters for level of anesthesia.

Page 1 of 4
 SOP-4 Tricaine Anesthetic Preparation
August 19, 2016 (Version 1)

S.O. P- 4 Synopsis
Tricaine-S Methanesulfonate Anesthetic Solution Preparation

Name of Biola University Zebrafish Lab

Institution/sponsor:

Active Spring water or conditioned water 48 ml, ethyl-amino benzoate (Tricaine-S

Chemicals/Ingredients: Methanesulfonate 0.2 g, NaOH 0.2 g

Tricaine-S solution is administered via immersion in solution. The TMS/MS-222 (Tricaine-S) is

absorbed through the skin and the gills and enters the bloodstream. Once in the bloodstream it is spread
throughout the body and ultimatley the brain, where the disruption of Sodium channels occurs.
MS-222/TMS is utilized to depress zebrafish nervous system resulting in loss of voluntary movement
and sensory perception. For this reason it is preferred during lab procedures to reduce stress on the fish
from being handled or during invasive procedures. Tricaine-S solution should always be used in a
purposeful and humane manner.

4.1 Chemistry
TMS is a white odorless crystalline powder that resembles a confectioner’s sugar consistency. It
is easily airborne and causes distress to the mucosal membranes when inhaled (careful when
handling). Tricaine-S is very soluble in water due to its polar properties. The drastic drop in PH
renders the solution for the fish to be far too acidic and therefore NaOH is utilized to buffer the
solution to a level that will be safe for the fish. The color of the solution may change to yellow
or brown when exposed to light. This does not negate the effects of sedation but some factors to
consider are temperature, hardness of water and PH. In cold water the effects of the Tricaine-S
are much less effective as in soft water and in low PH solutions. In low PH solutions the
Nitrogen containing portion of TMS is positively charged and has reduced permeability across
the gill membrane disabling its effects on the Sodium channels. Once in the bloodstream TMS is
metabolized by an acetylation reaction and are released via the gills (non-polar). Remaining
TMS is processed and released by the kidneys (polar) (Kathleen M. Carter, 2011).

Page 2 of 4
 SOP-4 Tricaine Anesthetic Preparation
August 19, 2016 (Version 1)

S.O. P- 4 Synopsis
Tricaine-S Methanesulfonate Anesthetic Solution Preparation

Name of Biola University Zebrafish Lab

Institution/sponsor:

Active Spring water or conditioned water 48 ml, ethyl-amino benzoate (Tricaine-S

Chemicals/Ingredients: Methanesulfonate 0.2 g, NaOH 0.2 g

4.2 Brief Legalities

In the United States, TMS is the only legal anesthetic for use on a limited number of food fish (FDA
2006). The limitations on TMS use with food fish include a 21-day withdrawal period before harvesting,
and use is restricted to the families Ictaluridae (catfishes), Salmonidae (salmon, trout, char, whitefish, and
grayling), Esocidae (pike and pickerel), and Percidae (perch, walleye, ruffe, and darters) (FDA 2006).
For other species, the drug should be limited to hatchery or laboratory applications in which fish will not
be released into the wild or consumed (Kathleen M. Carter, 2011).
4.3 Procedure
Volume:
Measure 48 mls of tap water/ conditioned fish water (from fish tank/housing). Pour into larger container
to further mix in solids.

Weigh:
Accurately (0.21-0.23 reading) weigh approximately 0.2 grams of Tricaine-S powder.
Accurately weigh approximately 0.2 grams of NaOH

Dissolve:

Mix in pre-weighed Tricaine powder to water container. Swirl solution to ensure large clumps or
discrepencies are dissolved well.

PH:

Use PH strips provided to check the PH of the Tricaine/water solution (should be around 3).

Buffer:

Mix in pre-weighed NaOH powder and swirl solution until fully visually dissolved. PH test the solution
prior to use on the fish. Ensure within 6.5-7.3 range for safe use with zebrafish.

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 SOP-4 Tricaine Anesthetic Preparation
August 19, 2016 (Version 1)

References
Kathleen M. Carter, C. M. (2011). A review of Tricaine Methanesulfonate for Anesthesia of Fish. Reviews
in Fish Biology and Fisheries, 21, 51.

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