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Haythorne’s Trichrome for Muscle and Collagen

Haythorne's Trichrome

for Muscle and Collagen



  • Böhmer’s alum hematoxylin
  • Solution A
    Orange G0.8g
    Ferric ammonium sulphate5g
    Hydrochloric acid0.06mL
    Ethanol, 95%4mL
    Distilled water100mL

    Dissolve the iron alum into 25 mL of the water.

    Combine the remaining ingredients.

    Mix the two solutions together and filter.

  • Solution B
    Acid fuchsin1g
    Distilled water100mL
  • Solution C
    Aniline blue2.5g
    Orange G2.5g
    Phosphomolybdic acidtosaturation
    Distilled water100mL

Tissue Sample

Zenker fixation was recommended, and 5µ paraffin sections would be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixation could probably be used with secondary fixation of sections in Bouin’s fluid.


  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Böhmer’s hematoxylin for 30 minutes.
  3. Place into solution A for 2 minutes.
  4. Wash with water for 5 minutes.
  5. Place into solution B for 3 minutes.
  6. Blot.
  7. Place into solution C for 20 minutes.
  8. Blot.
  9. Rinse quickly with 95% ethanol.
  10. Dehydrate and differentiate with absolute ethanol.
  11. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  reddish black
  • Erythrocytes  –  orange
  • Keratin  –  orange
  • Cytoplasm  –  red
  • Collagen  –  blue
  • Cartilage  –  blue


  • The iron alum in solution A probably serves to mordant the Böhmer’s hematoxylin, converting it into an iron hematoxylin, thus making the nuclear stain acid resistant.
  • Böhmer’s stain is an obsolete alum hematoxylin and a more modern progressive hemalum, such as Mayer’s, may be suitable.
  • Alternatively, Böhmer’s hematoxylin could probably be replaced with an acid resistant nuclear stain such as the celestine blue hemalum sequence, or Weigert’s iron hematoxylin. If so, the iron alum in solution A would likely be redundant (and the method would no longer be Haythorne’s).

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.
    Haythorne, (1916)
    Bulletin of the International Association of Medical Museums
    and Journal of Technical Methods., v. 6, p. 61
    Montreal, Que. Canada & Washington, DC, USA