Lendrum & McFarlane's Trichrome
for Muscle and Collagen
Materials
- Celestine blue-hemalum
- Solution A
Material Amount Picric acid 1 g Orange G 0.2 g Ethanol, 95% 80 mL Water 20 mL - Solution B
Material Amount 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
Material Amount Acetic acid, glacial 1 mL Distilled water 99 mL - Solution D
Material Amount Phosphomolybdic acid 1 g Distilled water 100 mL - Solution E
Material Amount for Variation I Amount for Variation 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.
Protocol
- Bring sections to water via xylene and ethanol.
- Stain nuclei with the celestine blue-hemalum sequence.
- Rinse well with water.
- Place into solution A for 2 minutes to overnight.
- Rinse with water.
- Place into solution B for 1-5 minutes.
- Rinse with solution C.
- Place into solution D until collagen is almost decolorized.
- Place into solution E for 2-10 minutes.
- Rinse with solution C.
- Return to solution E for 3-5 minutes.
- Dehydrate with absolute ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – blue
- Erythrocytes – yellow
- Cytoplasm – red
- Collagen – blue or green
Notes
- 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.
- Solution A calls for a weight of 1 gram of (dry) picric acid. An equivalent solution using a saturated ethanolic solution would be:
Material Amount Picric acid, sat. ethanolic 12 mL Orange G 0.2 g Ethanol, 95% 68 mL Water 20 mL
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- 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
