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Thomas’ Iron Hematoxylin

Thomas' Iron Hematoxylin



Solution A

Hematoxylin2.5 gDye
Dioxane40 mLSolvent
Hydrogen peroxide1 mLoxidizer

Solution B

Ferric ammonium sulfate3 gMordant
Ferric chloride1.5 gMordant
Ferrous chloride6 gMordant
Distilled water60 mLSolvent
Acetic acid6 mLAcidifier

Compounding Procedure

  1. Dissolve the hematoxylin into the dioxane.
  2. Add the peroxide.
  3. Add the acetic acid to the water.
  4. Dissolve the iron salts into the dilute acetic acid.
  5. Filter the solution of iron salts into the hematoxylin solution.


  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Wash well in running tap water to blue.
  4. Rinse with distilled water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained


  • The solution is likely stable for some time.
  • Dioxane is no longer used in histotechnology for safety reasons.The method should be considered obsolete.
  • The solution is progressive, and the staining time should be determined by trial.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.


  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Thomas, (1943)
    Bulletin d’histologie appliquée à la physiologie et à la pathologie et de technique microscopique, v. 20, p. 212.