Haythorne's Trichrome
for Muscle and Collagen

Solutions

Böhmer's alum hematoxylin

Solution A
Orange G	0.8	g
Ferric ammonium sulphate	5	g
Hydrochloric acid	0.06	mL
Ethanol, 95%	4	mL
Distilled water	100	mL

Dissolve the iron alum into 25 mL of the water.

Combine the remaining ingredients.

Mix the two solutions together and filter.

Solution B
Acid fuchsin	1	g
Distilled water	100	mL

Solution C
Aniline blue	2.5	g
Orange G	2.5	g
Phosphomolybdic acid	to	saturation
Distilled water	100	mL

Tissue sample
Zenker fixation was recommended, and 5µ paraffin sections would be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation.
Formalin fixation could probably be used with secondary fixation of sections in Bouin's fluid.

Method

1. Bring sections to water via xylene and ethanol.
2. Stain nuclei with Böhmer's hematoxylin for 30 minutes.
3. Place into solution A for 2 minutes.
4. Wash with water for 5 minutes.
5. Place into solution B for 3 minutes.
6. Blot.
7. Place into solution C for 20 minutes.
8. Blot.
9. Rinse quickly with 95% ethanol.
10. Dehydrate and differentiate with absolute ethanol.
11. Clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  reddish black
• Erythrocytes  –  orange
• Keratin  –  orange
• Cytoplasm  –  red
• Collagen  –  blue
• Cartilage  –  blue

Notes

1. The iron alum in solution A probably serves to mordant the Böhmer's hematoxylin, converting it into an iron hematoxylin, thus making the nuclear stain acid resistant.
2. As Böhmer's is an alum hematoxylin, possibly a more modern progressive hemalum, such as Mayer's, may be suitable.
3. Alternatively, Böhmer's hematoxylin could probably be replaced with an acid resistant nuclear stain such as the celestine blue hemalum sequence, or Weigert's iron hematoxylin. If so, the iron alum in solution A would likely be redundant (and the method would no longer be Haythorne's).

 

Reference

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
  Citing:–
    Haythorne, (1916)
    Bulletin of the International Association of Medical Museums
    and Journal of Technical Methods., v. 6, p. 61
    Montreal, Que. Canada & Washington, DC, USA

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