Watson's Alum Hematoxylin
Materials
| Material | 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, then add 120 mL)
- If using chloramine T or barium hydroxide, add to the solution.
Protocol
- Bring sections to water with xylene and ethanol.
- Place into the staining solution for an appropriate time.
- Rinse well with water.
- Differentiate with acid ethanol if necessary.
- Rinse with water and blue.
- Rinse well with water.
- Counterstain if desired.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – blue
- Background – as counterstain or unstained
Notes
- Use only one of the oxidants named.
- 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.
- Gray gives a similar formula for Watson’s hemalum with potassium permanganate, but specifies only 0.6 grams alum. This may be an error.
- The staining time should be determined by trial.
- Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
- 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.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- 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.
