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McFarlane’s Trichrome 2nd Variant for Muscle and Collagen

McFarlane's Trichrome 2nd Variant

for Muscle and Collagen



  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    Picric acid0.2g
    Acid fuchsin0.8g
    Acetic acid, glacial2mL
    Distilled water98mL
  • Solution B
    Acetic acid, glacial2mL
    Distilled water98mL
  • Solution C
    Picric acid1g
    Phosphotungstic acid10g
    Ethanol, 95%40mL
    Distilled water60mL
  • Solution D
    Aniline blue2.5mL
    Acetic acid, glacial2.5g
    Distilled water97.5mL
  • Solution E
    Picric acid0.25g
    Phosphotungstic acid2.5g
    Ethanol, 95%10mL
    Distilled water90mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.


  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert’s iron hematoxylin or equivalent.
  3. Place into solution A for 5 minute.
  4. Rinse with solution B.
  5. Place into solution C for 5 minutes.
  6. Rinse with distilled water.
  7. Place into solution D for 5-10 minutes.
  8. Rinse with solution B.
  9. Place into solution E for 5 minutes.
  10. Wash with solution B.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Cytoplasm  –  red
  • Muscle  –  red
  • Collagen  –  blue


  • McFarlane also has another multi-step method and a one step method.
  • Solutions A, C and E require dry weights of picric acid. These could be provided from saturated aqueous (1 g in 78 mL) or ethanolic (1 g in 12 mL) solutions. Alternate formulas are:

    Solution A

    Picric acid, sat. alc.16mL
    Distilled water82mL
    Acetic acid, glacial2mL
    Acid fuchsin0.8g

    Solution C

    Picric acid, sat. alc.12mL
    Distilled water60mL
    Ethanol, 95%28mL
    Phosphotungstic acid10g

    Solution E

    Picric acid, sat. alc.20mL
    Distilled water70mL
    Ethanol, 95%10mL
    Phosphotungstic acid2.5g

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.
    McFarlane, (1944)
    Stain Technology, v. 19, pp. 29