Ehrlich's Alum Hematoxylin
Materials
| Material | Amount | Function |
|---|---|---|
| Hematoxylin | 6 g | Dye |
| 95% ethanol | 300 mL | Solvent |
| Potassium alum | excess | Mordant |
| Distilled water | 300 mL | Solvent |
| Glycerol | 300 mL | Stabiliser |
| Glacial acetic acid | 30 mL | Acidifier |
Compounding procedures
- Dissolve the hematoxylin in the ethanol mixed with acetic acid.
- Dissolve the alum in the water mixed with glycerol in an oversized container.
- Add the hematoxylin solution to the alum solution.
- Plug the container loosely with cotton wool.
- Ripen by leaving in a warm, sunlit place for several weeks.
- When sufficiently ripened, store tightly stoppered in a cool, dark place.
- The solution is stable for years.
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
- Gray gives 7 grams hematoxylin, and specifies ammonium alum.
- The alum should be added to excess. This should be about 50 grams, but enough should be added to ensure undissolved alum is present.
- This is a strongly staining, regressive formula. The staining time should be determined by trial. Usually, 20 minutes is adequate.
- As with many strong alum hematoxylin solutions, cartilage, cement lines and mucin may stain blue.
- The solution may be chemically ripened by adding 0.5g sodium iodate, but chemicallly ripened solutions are inferior in longevity.
- 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
- Culling, C.F.A., Allison, R.T. and Barr, W.T.
Cellular Pathology Technique, Ed.4.
Butterworth, London, UK. - Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK. - Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK. - Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Ehrlich, (1886)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v. 3, p. 150.
Leipzig.
