What is Masson’s Trichrome Stain used for?
It is used to differentiate between collagen and smooth muscle in tumors and to detect increased collagen in diseases like cirrhosis.
What types of biopsies is Masson’s Trichrome Stain routinely used for?
It is commonly used for liver and kidney biopsies.
What is the principle behind Masson’s Trichrome Stain?
It uses three dyes to selectively stain muscle, collagen fibers, fibrin, and erythrocytes based on tissue porosity and molecular size.
What are the three dyes used in Masson’s Trichrome Stain?
The dyes are Biebrich Scarlet, Weigert’s Iron Hematoxylin, and Aniline Blue.
What is the role of Biebrich Scarlet in the staining process?
It binds with acidophilic tissue components and helps differentiate between tissues.
What is the role of Aniline Blue in the staining process?
It binds with collagen, staining it blue.
What is the role of Weigert’s Iron Hematoxylin in the staining process?
It stains nuclei black.
What fixative is preferred for Masson’s Trichrome Stain?
Bouin’s fixative is preferred.
What are the components of Bouin’s fixative?
It contains saturated picric acid, formaldehyde, and glacial acetic acid.
Why is Bouin’s fixative considered hazardous?
It contains formaldehyde (a carcinogen) and picric acid (which can become explosive when dry).
What is the thickness of paraffin sections recommended for Masson’s Trichrome Stain?
Sections should be cut at 4 micrometers.
What equipment is needed for Masson’s Trichrome Stain?
Coplin jars, a 60°C oven or water bath, and a microwave are required.
How long should sections be mordanted in Bouin’s solution using the microwave method?
1 minute in the microwave, followed by standing for 15 minutes.
What is the conventional method for mordanting in Bouin’s solution?
Mordant at 60°C for 1 hour.
How long should sections be washed in running tap water after Bouin’s fixation?
5 minutes to remove picric acid.
How long should sections be stained with Weigert’s working hematoxylin?
10 minutes.
How long should sections be treated with Biebrich Scarlet?
5 minutes.
How long should sections be treated with phosphotungstic/phosphomolybdic acid?
10 minutes.
How long should sections be stained with Aniline Blue?
5 minutes.
What is the purpose of rinsing with 1% acetic acid?
It helps differentiate the stain and remove excess dye.
How is Bouin’s fixative prepared?
Mix 1500 ml saturated picric acid, 500 ml formaldehyde, and 100 ml glacial acetic acid.
How long is Bouin’s fixative stable?
It is stable for 2 years.
How is Biebrich Scarlet solution prepared?
Mix 2.7 gm Biebrich Scarlet, 0.3 gm Acid Fuchsin, 300 ml distilled water, and 3 ml glacial acetic acid.
How long is Biebrich Scarlet solution stable?
It is stable for 6 months.
How is Weigert’s Iron Hematoxylin Stock Solution A prepared?
Mix 5 gm Hematoxylin with 500 ml 95% alcohol.
How is Weigert’s Iron Hematoxylin Stock Solution B prepared?
Mix 20 ml 29% ferric chloride, 475 ml distilled water, and 5 ml hydrochloric acid.
How long is Weigert’s Iron Hematoxylin working solution stable?
It is stable for 3-4 days.
How is the phosphotungstic/phosphomolybdic acid solution prepared?
Mix 25 gm phosphotungstic acid, 25 gm phosphomolybdic acid, and 1000 ml distilled water.
How long is the phosphotungstic/phosphomolybdic acid solution stable?
It is stable for 6 months.
How is Aniline Blue solution prepared?
Mix 2.5 gm Aniline Blue, 100 ml distilled water, and 1 ml glacial acetic acid.
How long is Aniline Blue solution stable?
It is stable for 6 months.
How is 1% acetic acid prepared?
Mix 10 ml glacial acetic acid with 1000 ml distilled water.
How long is 1% acetic acid stable?
It is stable for 1 year.
What safety precautions should be taken when handling Bouin’s fixative?
Wear gloves, goggles, and a lab coat. Keep it under a fume hood.
Why is picric acid hazardous?
It is toxic by skin absorption and can become explosive when dry.
What are the hazards of phosphotungstic and phosphomolybdic acids?
They are skin and eye irritants and strong corrosives.
What are the hazards of glacial acetic acid?
It is a skin and eye irritant.
What PPE is required for Masson’s Trichrome Stain?
Gloves, goggles, and a lab coat.
Why should Bouin’s fixative be kept under a fume hood?
To avoid inhalation of formaldehyde fumes.
What should be done if Bouin’s fixative dries out?
Handle with extreme caution as picric acid can become explosive.
What color do nuclei stain with Masson’s Trichrome Stain?
Black.
What color do cytoplasm, muscle, and erythrocytes stain?
Red.
What color does collagen stain?
Blue.
Can Light Green be substituted for Aniline Blue?
Yes, but 5% phosphotungstic acid must be used instead of the standard solution.
What is the preferred collagen color for liver biopsies?
Light blue rather than dark blue.
What is the purpose of using phosphotungstic/phosphomolybdic acid?
It helps differentiate between tissue components by pulling red dye out of collagen.
What is the role of glacial acetic acid in the staining process?
It acts as a differentiating agent.
Why is distilled water used for rinsing?
To avoid contamination and ensure consistent staining.
What is the purpose of dehydrating and clearing the sections?
To prepare the tissue for mounting and coverslipping.
What is the shelf life of Weigert’s Iron Hematoxylin stock solutions?
Both Stock Solution A and B are stable for 1 year.
