McFarlane's Trichrome
1st Variant for Muscle and Collagen

Solutions

Weigert's iron hematoxylin or equivalent

Solution A
Picric acid	0.2	g
Acid fuchsin	1	g
Aniline blue	2	g
Phosphotungstic acid	1	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Solution B
Acetic acid, glacial	2	mL
Distilled water	100	mL

Solution C
Picric acid	0.25	g
Phosphotungstic acid	2.5	g
Ethanol, 95%	10	mL
Distilled water	90	mL

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.

Method

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. Rinse with distilled water.
6. Place into solution C until differentiated.
7. Rinse with distilled water.
8. Wash with solution B.
9. Dehydrate with ethanol.
10. Clear with xylene and mount with a resinous medium.

Expected results

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

Notes

1. McFarlane also published two other, multi-step, trichrome methods. One is simpler than the other.
2. Solutions A and C require dry weights of picric acid. This could be provided from a saturated aqueous solution (1 g in 78 mL). Alternate formulas are:–
    
   

   Solution A				Solution C
   Picric acid, sat. aqueous	16	mL		Picric acid, sat. aqueous	20	mL
   Distilled water	82	mL		Distilled water	70	mL
   Acetic acid, glacial	2	mL		Ethanol, 95%	10	mL
   Acid fuchsin	1	g		Phosphotungstic acid	2.5	g
   Aniline blue	2	g
   Phosphotungstic acid	1	g

 

Reference
Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
  Citing:–
    McFarlane, (1944)
    Stain Technology, v. 19, pp. 29

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