Alum Hematoxylin Variants

Ingredient Var I Var II Function
Hematoxylin 10 g 4 g Dye
Ammonium or potassium alum 50 g 50 g Mordant
Distilled water 1 L 1 L Solvent
95% ethanol 50 mL   Solvent
Mercuric oxide 5 g   Oxidant
Sodium iodate   0.3 g Oxidant
Glacial acetic acid 20 mL   Acidifier
Citric acid   1.5 g Acidifier
Chloral hydrate   75 g Antioxidant


Compounding procedure

Var I
Dissolve the dye into the ethanol, and the alum into the water.
Combine the solutions in an Erlenmeyer flask and slowly add the mercuric oxide
Reheat until the solution changes colour to a dark purple.
Remove from heat and cool rapidly by swirling the flask in cold water.
When cool, add the acetic acid.

Var II
Dissolve the alum into the water.
Add the hematoxylin, sodium iodate, citric acid and chloral hydrate in that order.
Filter before use.


  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for the specified time.
    • Var I for 10 minutes.
    • Var II for 8-10 seconds.
  3. Rinse well with water.
  4. Differentiate Var I with acid ethanol for a few seconds.
  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. Var I was recommended for double embedded tissues.
  2. The use of mercuric oxide as an oxidant is now deprecated due to its toxicity. Var I could be modified by using sodium iodate at about 0.3 grams instead.
  3. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
  4. 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.


Molnar, L. N.,
Modification of Harris hematoxylin for sections from tissue double embedded with nitrocellulose and paraffin.
Histologic, v 5, Nº 1, January, 1975

Molnar, L. N.,
Modification of Mayer's hematoxylin-eosin method.
Histologic, v 6, Nº 4, October, 1976



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