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Tricaine / MS-222 Dosing Calculator.

Calculate MS-222 working solutions for zebrafish sedation, anesthesia, and euthanasia. Computes powder and stock dilution recipes with sodium bicarbonate buffering.

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Validated2026-04-06
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Load example MS-222 Dosing data to see the full workflow

Configuration

Recommended: 100–200 mg/L

Dilute from stock solution

When to use

  • Preparing MS-222 working solutions for zebrafish sedation, anesthesia, or euthanasia
  • Calculating stock solution dilutions for specific experimental volumes
  • Determining sodium bicarbonate buffer amounts for pH neutralization
  • Preparing anesthetic solutions for larvae vs. adult zebrafish

Do not use for

  • As a substitute for IACUC-approved protocols — always follow your institutional SOP
  • For non-aquatic species — MS-222 dosing differs for amphibians and other taxa
  • For determining anesthesia depth — monitor fish behavior directly

Always buffer with sodium bicarbonate

Unbuffered MS-222 drops water pH to 3–4, causing gill epithelial damage and erratic recovery. The standard 2:1 (NaHCO₃:tricaine) ratio brings pH to 7.0–7.4. Always verify pH with a meter or strips before use.

Temperature affects induction time

Fish at 28.5°C are anesthetized faster than fish at 24°C at the same MS-222 concentration. If your facility runs cooler than standard, induction may take longer — do not compensate by increasing concentration without IACUC consultation.

Larvae are more sensitive

Zebrafish larvae (<14 dpf) require lower concentrations (50–100 mg/L for anesthesia vs. 100–200 mg/L for adults). Chorionated embryos are partially protected, but dechorionated embryos are fully exposed.

Check stock solution freshness

Degraded MS-222 turns yellow-brown and loses potency. Fresh stock should be colorless to very pale yellow. If your induction times are increasing, prepare fresh stock before adjusting concentration.

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Method

Direct mass-balance calculation: tricaine mass = concentration ×\times volume. Buffer mass = tricaine mass ×\times 2 (standard 2:1 NaHCO₃:tricaine ratio). Stock dilutions use C₁V₁ = C₂V₂. Concentration ranges per AVMA Guidelines (2020) and Collymore et al. (2014).

2

Validated

Last validated 2026-04-06. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

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How to cite

How to Cite

ConductScience Tricaine / MS-222 Dosing Calculator (v1.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/ms222-dosing-calculator

Collymore C et al. Efficacy and safety of 5 anesthetics in adult zebrafish. JAALAS. 2014;53(2):198–203.

AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. AVMA, Schaumburg, IL.

MS-222 Pharmacology in Zebrafish

Tricaine methanesulfonate (ethyl 3-aminobenzoate methanesulfonate, MS-222, Tricaine-S, TMS) is the most widely used anesthetic for zebrafish and other aquatic vertebrates. It acts by blocking voltage-gated sodium channels in neurons, producing reversible loss of sensation and motor function.

Stages of anesthesia in zebrafish: - Stage I (light sedation): Reduced reaction to stimuli, normal opercular rate - Stage II (deep sedation): Decreased opercular rate, reduced equilibrium - Stage III (surgical anesthesia): Loss of equilibrium, no response to tail pinch, slow opercular movement - Stage IV (medullary collapse): Cessation of opercular movement — euthanasia threshold

Recovery is achieved by transferring fish to fresh system water. Recovery time depends on concentration, exposure duration, and water temperature.

Safety and Regulatory Notes

MS-222 is a DEA-exempt substance but is regulated for use in food fish (21-day withdrawal period per FDA). For zebrafish research, the primary regulatory considerations are:

  • IACUC approval is required for all anesthesia and euthanasia protocols
  • pH buffering is mandatory — unbuffered MS-222 causes gill damage
  • Euthanasia confirmation requires 10 min post-cessation of opercular movement (AVMA Guidelines 2020)
  • Waste disposal — follow institutional chemical waste procedures; do not pour down drain
  • PPE — wear gloves when handling; MS-222 can cause skin sensitization with repeated exposure

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