Many recipes are available for this anesthetic. Please use the instructions below for preparing Avertin solutions to avoid problems due to endotoxins, soap residues, or breakdown products that may be toxic, and result in anesthetic deterioration over time. Avertin has been reported to cause peritonitis, abdominal adhesions, ileus, and even death. (1,2,3)
Avertin is not recommended for repeated anesthesia; however, the IACUC will consider protocols that request administering 2 independent full doses per animal when adequate scientific justification is provided.
Avertin (2.5% tribromoethanol)
The following recipe makes a 2.5% solution of Avertin. It is recommended that Avertin be mixed using acid washed glass which has been rinsed in distilled water. Glassware washed in soap may leave a residue that may be toxic.
- 2,2,2-tribromoethanol (99%): 10 g, i.e., Acros Organics 421430100 (others sources available)
- Tert-amyl alcohol, reagent grade: 1 liter, i.e., Fisher A730-1
Preparation of stock solution
- Add 6.2 ml tert-amyl alcohol to 10 g Avertin (2,2,2-tribromoethanol) [Scale up if a greater volume is required]. The bottle in which the Avertin arrives is convenient. Concentration of stock solution is 1.61 g/ml.
- Drop in a stir bar and stir on a magnetic stirrer until Avertin is completely dissolved. This will probably take overnight.
- Keep the stock in the dark bottle and tightly capped. Store at room temp. The Avertin stock is photosensitive and hydroscopic. The photo-oxidation products are toxic. Layering dry nitrogen gas or Freon from an aerosol duster over the solution is an excellent idea. Stock solution is stable for 6-12 months. Yellow discoloration indicates presence of toxic products. Indicate on the bottle the date the avertin was dissolved.
Preparation of working strength solution (2.5%)
- Ensure that glassware and cylinders are clean. Pre-treating a 50 ml graduated cylinder with 10% HCl to remove detergent residue and thoroughly rinsing the cylinder with ddH2O is recommended.
- Add 49.2 ml pre-warmed tissue culture grade ddH2O to the cylinder (containing a stir bar). Then add drop-wise with constant stirring 0.78 ml of the Avertin stock solution. Seal cylinder with Parafilm and completely wrap with aluminum foil to exclude light.
- Stir slowly on low heat overnight to dissolve Avertin stock solution.
- Filter the working solution through a 0.2 micrometer filter into a dark glass bottle.
- Store the working solution at 4°C, except when using. Label and date the bottle on day of preparation. The working solution is stable for at least 6 months. After six months, any unused solution should be appropriately discarded following local, state or federal regulations.
After the working stock is prepared, it is recommended that a titration be performed to measure the depth of anesthesia. Ranges from 0.300-0.400 ml have been used in 18-21 g mice. Generally 0.35 ml injected intraperitoneally (i.p.) to anesthetize a young adult (18 g) mouse will produce approximately 30 minutes of anesthesia. Adjust volume depending on size of mouse. Test level of anesthesia by pinch reflex. Anesthetic recovery is to be adequately monitored by the investigator.
1Green, C J, 1975. Neuroleptanalgesic drug combinations in the anesthetic management of small laboratory animals. Lab Animals. 9:161-178.
2Papaioannou, V, and Fox, J. 1993. Efficacy of Tribromoethanol Anesthesia in Mice. Lab Animal Science. 43(2):189-192.
3Zeller, W, Meier, G, Burki, K, and Panoussis B. 1998. Adverse effects of tribromoethanol as used in the production of transgenic mice. Lab Animals. 32:407-413.
4Sangster, M, Sparer, T. 2009. Personal Communication – UTK.