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Lendrum & McFarlane’s Trichrome for Muscle and Collagen

Lendrum & McFarlane's Trichrome

for Muscle and Collagen



  • Celestine blue-hemalum
  • Solution A
    Picric acid1g
    Orange G0.2g
    Ethanol, 95%80mL
  • Solution B
    Acid fuchsin0.5g
    Ponceau 2R0.5g
    Sodium sulphate0.25g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Solution C
    Acetic acid, glacial1mL
    Distilled water99mL
  • Solution D
    Phosphomolybdic acid1g
    Distilled water100mL
  • Solution E
    MaterialAmount for Variation IAmount for Variation II
    Aniline blue2g
    Fast green FCF2g
    Acetic acid, glacial1mL1mL
    Distilled water100mL100mL

Tissue Sample

Most trichrome stains benefit from picric acid or mercuric chloride fixation. 5µ paraffin sections of neutral buffered formalin fixed tissue would probably be suitable, although secondary fixation of sections in Bouin’s fluid overnight or at 56°C for an hour would undoubtedly enhance staining.


  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with the celestine blue-hemalum sequence.
  3. Rinse well with water.
  4. Place into solution A for 2 minutes to overnight.
  5. Rinse with water.
  6. Place into solution B for 1-5 minutes.
  7. Rinse with solution C.
  8. Place into solution D until collagen is almost decolorized.
  9. Place into solution E for 2-10 minutes.
  10. Rinse with solution C.
  11. Return to solution E for 3-5 minutes.
  12. Dehydrate with absolute ethanol.
  13. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Erythrocytes  –  yellow
  • Cytoplasm  –  red
  • Collagen  –  blue or green


  • The method appears to call for solution A to be applied for 2 minutes to overnight, although the instructions in Gray are not completely clear. It should be noted that this solution contains sufficient picric acid to act as a fixative as well as to stain erythrocytes, and overnight treatment might function partly to bring about some secondary fixation. For tissue well fixed with Bouin or a mercuric chloride fixative, 2 minutes should be adequate.
  • Solution A calls for a weight of 1 gram of (dry) picric acid. An equivalent solution using a saturated ethanolic solution would be:
    Picric acid, sat. ethanolic12mL
    Orange G0.2g
    Ethanol, 95%68mL

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.
    Lendrum and McFarlane, (1940)
    Journal of Pathology and Bacteriology, v. 50, pp. 381