Alizarin Red S and Toluidine Blue O

EMS26205

Components:

Alizarin Red S 0.02% Aqueous

Toluidine Blue O 0.25%

Ammonium Alcohol

Potassium Hydroxide

Sulphuric Acid Alcohol

Potassium Hydroxide 4% Aqueous

Fixation:

At least 1 week in 10% Formalin after evisceration and washing. (Evisceration is not necessary for museum specimen)

Staining Procedures:

1. Wash the specimen in 250ml of Ammonium Alcohol. For 24 hours.
2. Stain in Toluidine Blue Solution, 0.25% for 1 week.
3. Harden and de-stain for 72 hours in 4 changes of 95% alcohol.
4. Macerate in several changes of Potassium Hydroxide, 2% for 5 – 7 days, depending on the size of animal. This step is facilitated exposure to sunlight or ultraviolet light.
5. Transfer to Working* Alizarin Red S solution for approximately 24 hours.
   1. * Prepare the Working Stain by mixing equal parts of Alizarin Red S, 0.02% and Potassium Hydroxide, 4%. Prepare fresh.
      If specimen has been insufficiently macerated the soft will appear slightly attained. They may be quickly de-stained in Sulphuric Acid-Alcohol.
6. Dehydrate through 3 changes of cellosolve, 6 hours each. Reduce the time for small embryos. OPTIONAL: One may substitute the alcohol series, 50%, 80% and 90% followed by 3 changes of benzene.
7. Clear by transferring to solution for 25%, 50%, and 75% methyl salicylate in cellosolve for 24 hours each. Then store in methyl salicylate.
   Modification by using glycerin for clearing: Omit step 6 and transfer directly from Working Alizarin Red S solution into a series of 50%, 70% and 80% glycerin solution for 24 hours each. Store in glycerin.

Stain Results:

Note: The intensity of the stains serves to indicate the relative amounts of ossification and chrondogeneis, which may be taken place. The bone cartilage may be stained separated by omitting step 2 for bone or step 5 for cartilage.

References:

Dawson, A.B., Stain Tech., 1:123 – 4, 1926.

Williams, T.W., Stain Techn., 16:23-5, 1941.

Clark, G., (ed)., Staining Procedures, 3rd ed., Williams & Wilkins, Baltimore, p. 139, c. 1973.