Ehrlich's
Alum Hematoxylin

Ingredient	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 procedure
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.

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. Gray gives 7 grams hematoxylin, and specifies ammonium alum.
2. The alum should be added to excess. This should be about 50 grams, but enough should be added to ensure undissolved alum is present.
3. This is a strongly staining, regressive formula. The staining time should be determined by trial. Usually, 20 minutes is adequate.
4. As with many strong alum hematoxylin solutions, cartilage, cement lines and mucin may stain blue.
5. The solution may be chemically ripened by adding 0.5g sodium iodate, but chemicallly ripened solutions are inferior in longevity.
6. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
7. 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
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.