Milligan's trichrome
for Muscle and Collagen


Solution A
Potassium dichromate 2.25 g
Hydrochloric acid, conc 2.25 mL
Ethanol, 95% 25 mL
Distilled water 75 mL
Solution B
Acid fuchsin 0.1 g
Distilled water 100 mL
Solution C
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution D
Orange G 2 g
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution E
Acetic acid, glacial 1 mL
Distilled water 100 mL
Solution F
Fast green FCF 0.1 g
Acetic acid, glacial 0.2 mL
Distilled water 100 mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed were specified. Other fixatives may 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. Place into solution A for 5 minute.
  3. Rinse with distilled water.
  4. Place into solution B for 5 minutes.
  5. Rinse quickly with distilled water.
  6. Place into solution C for 1-5 minutes.
  7. Rinse with distilled water.
  8. Place into solution D for 5-10 minutes.
  9. Rinse with distilled water.
  10. Place into solution E for 2 minutes.
  11. Place into solution F for 5-10 minutes.
  12. Place into solution E for 3 minutes.
  13. Dehydrate with ethanol.
  14. Clear with xylene and mount with a resinous medium.

Expected results


  1. Aniline blue may be substituted for fast green FCF at the same concentration. Collagen would then be blue.


Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
    Milligan, (1946)
    Technical Bulletin, v. 7, pp.57




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