Lendrum and McFarlane's
Trichrome for Muscle and Collagen

Solutions

Celestine blue-hemalum

Solution A
Picric acid	1	g
Orange G	0.2	g
Ethanol, 95%	80	mL
Water	20	mL

Solution B
Acid fuchsin	0.5	g
Ponceau 2R	0.5	g
Sodium sulphate	0.25	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Solution C
Acetic acid, glacial	1	mL
Distilled water	99	mL

Solution D
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution E		var I			var II
Aniline blue		2	g		–
Fast green FCF		–			2	g
Acetic acid, glacial		1	mL		1	mL
Distilled water		100	mL		100	mL

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.

Method

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 decolorised.
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

Notes

1. 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.
2. Solution A calls for a weight of 1 gram of (dry) picric acid. An equivalent solution using a saturated ethanolic solution would be:–

   Solution A
   Picric acid, sat. ethanolic	12	mL
   Orange G	0.2	g
   Ethanol, 95%	68	mL
   Water	20	mL

 

Reference

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
  Citing:–
    Lendrum and Mcfarlane, (1940)
    Journal of Pathology and Bacteriology, v. 50, pp. 381
     London, UK.

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