De Groot's Alum Hematoxylin

Ingredient Amount Function
Hematoxylin 2 g Dye
Ammonium alum 22 g Mordant
Distilled water 270 mL Solvent
95% ethanol 650 mL Solvent
Glycerol 80 mL Stabiliser
Hydrogen peroxide 7.5 mL Oxidant
Potassium ferricyanide 0.8 g
Calcium chloride 15 g
Sodium bromide 7.5 g


Compounding procedure
Mix the ethanol, water and glycerol to make the solvent.
Add the peroxide to 15 mL of the solvent.
Add the hematoxylin, and dissolve.
Dissolve the calcium chloride and sodium bromide in 250 mL of the solvent.
Mix with the hematoxylin solution.
Add half the alum, and dissolve.
Dissolve the potassium ferricyanide in 400 mL of the solvent.
Add to the hematoxylin solution.
Dissolve the remaining Alum in the remaining solvent.
Add to the hematoxylin solution.


  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


  1. Both hydrogen peroxide and potassium ferricyanide are oxidising agents. However, it is not clear if potassium ferricyanide is present for this reason.
  2. The strength of hydrogen peroxide is not specified, but the commonest laboratory strength is 30 vols.
  3. Although calcium can mordant hematoxylin, it is not clear if it is present for that reason.
  4. The purpose of the sodium bromide is not clear. It may be present as a preservative, similar to chloral hydrate in some other formulas.
  5. The staining time should be determined by trial.
  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.


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




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