Bensley's trichrome
for Muscle and Collagen

Solutions

Solution A
Acid fuchsin	to	saturation
Aniline water	100	mL

Add about 20 g dye to the aniline water.

Shake intermittently for 48 hours. Filter.

Solution B
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution C
Orange G	2	g
Aniline blue	0.5	g
Distilled water	100	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. Place into solution A for 10 minute.
3. Rinse with distilled water.
4. Place into solution B for 10 minutes.
5. Rinse quickly with distilled water.
6. Place into solution C for 60 minutes.
7. Rinse with 95% ethanol until clouds of dye cease being extracted.
8. Dehydrate with ethanol.
9. Clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  red
• Erythrocytes  –  orange
• Cytoplasm  –  red
• Collagen  –  blue

Notes

1. Solution A is Altmann's acid fuchsin, originally formulated for staining mitochondria.
2. This method does not specify an acid resistant nuclear stain. Doing so before staining with solution A may improve the technique.

Reference

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
  Citing:–
    Mallory, F. B., (1938),
    Pathological technique.
    Saunders, Philadelphia, Pa, USA