Watson's
Alum Hematoxylin

Ingredient	Amount	Function
Hematoxylin	6 g	Dye
Ammonium alum	6 g	Mordant
Distilled water	300 mL	Solvent
100% ethanol	300 mL	Solvent
Glycerol	300 mL	Stabiliser
Glacial acetic acid	30 mL	Acidifier
Potassium permanganate or Chloramine T or Barium hydroxide	0.3 g 3.9 g 6 g	Oxidants

 

Compounding procedure
Dissolve the alum in the water.
If using potassium permanganate add to the alum solution.
Dissolve the hematoxylin in ethanol.
Combine the two solutions.
Add the glycerol.
Add the acetic acid (30 mL, unless using barium hydroxide when add 120 mL)
If using chloramine T or barium hydroxide add to the solution.

Method

1. Bring sections to water with xylene and ethanol.
2. Place into the staining solution for an appropriate time.
3. Rinse well with water.
4. Differentiate with acid ethanol if necessary.
5. Rinse with water and blue.
6. Rinse well with water.
7. Counterstain if desired.
8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  blue
• Background  –  as counterstain or unstained

Notes

1. Use only one of the oxidants named.
2. This is a modification of Ehrlich's hematoxylin, and is said to be as effective. It was designed to be used immediately after preparation, eliminating the months required for atmospheric oxidation.
3. Gray gives a similar formula for Watson's hemalum with potassium permanganate, but specifies only 0.6 grams alum. This may be an error.
4. The staining time should be determined by trial.
5. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
6. Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

 

Reference
Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist's Vade-Mecum. 11 ed.,
Churchill, London, UK.
  Citing:–
    Watson, (1943)
    Journal of the Royal Microscopical Society, v. 63, p. 20

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.