Slidders’ Fuchsin-Miller for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Slidders’ Fuchsin-Miller

for Fibrin

steps

materials

print

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Fuchsin
Material Amount
Acid fuchsin 1 g
Acetic acid, glacial 2.5 mL
Distilled water 100 mL
Miller
Material Amount
Milling yellow 3G 2.5 g
2-ethoxy-ethanol 100 mL
Phosphotungstic acid
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL

Material	Amount
Acid fuchsin	1	g
Acetic acid, glacial	2.5	mL
Distilled water	100	mL

Material	Amount
Milling yellow 3G	2.5	g
2-ethoxy-ethanol	100	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate or B5 overnight. Paraffin process overnight. Overnight formalin fixation and paraffin processing can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
If mercury fixed, remove with the iodine-thiosulphate sequence.
If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
Rinse well with distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in fuchsin for 10 minutes.
Rinse with distilled water.
Differentiate with phosphotungstic acid for 5 minutes.
Rinse well with distilled water.
Blot.
Rinse well with cellosolve.
Place into milling yellow until fibrin is red and tissues are yellow.
This step takes from ½ – 4 hours.
Rinse briefly with 1% aqueous acetic acid.
Rinse with cellosolve.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – red
Other tissue – yellow
Nuclei – black

Notes

It is very important that the milling yellow solution be completely anhydrous. Even small amounts of water or other solvents can cause problems with displacement and incomplete removal of red dye. For that reason, step 12 should be thorough and the milling yellow should be used in a Coplin jar with a lid sealed with tape.
A well stained section would show red fibrin only, muscle and erythrocytes being yellow. With poorly fixed material, both erythrocytes and muscle fibres may resist displacement of the red dye and they may have some residual red colouring. Sometimes this may be overcome by prestaining either with saturated picric acid in absolute ethanol or the MSB martius yellow solution for two minutes immediately before step 7, but a better resolution is correct fixation and processing.
Some intracellular materials may stain red, such as Paneth cell granules.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Martius, Scarlet and Blue (MSB) for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Martius, Scarlet and Blue (MSB)

for Fibrin

steps

materials

print

The MSB (Martius, Scarlet and Blue) method for fibrin is a reliable technique. It is more automatic than other methods, i.e. it is less dependent on skill and experience and is consequently suitable for a routine laboratory. Overnight mercuric chloride fixation (formol sublimate or B5) is preferred, followed by overnight paraffin processing, although formalin fixed, paraffin embedded material can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections, as for the Picro-Mallory.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Martius
Material Amount
Martius yellow 0.5 g
Phosphotungstic acid 2 g
Ethanol, 95% 100 mL
Scarlet
Material Amount
Crystal scarlet 1 g
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Blue
Material Amount
Methyl blue 0.5 g
Acetic acid, glacial 1 mL
Distilled water 98 mL
Phosphotungstic acid
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL

Material	Amount
Martius yellow	0.5	g
Phosphotungstic acid	2	g
Ethanol, 95%	100	mL

Material	Amount
Crystal scarlet	1	g
Acetic acid, glacial	2.5	mL
Distilled water	97.5	mL

Material	Amount
Methyl blue	0.5	g
Acetic acid, glacial	1	mL
Distilled water	98	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Overnight formalin fixation is usually satisfactory, but avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
If mercury fixed, remove with the iodine-thiosulphate sequence.
If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
Rinse well in distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in martius yellow for 2 minutes.
Rinse with distilled water.
Place in crystal scarlet for 10 minutes.
Differentiate with phosphotungstic acid until only fibrin is red (up to 10 minutes).
Place in methyl blue until collagen is blue (up to 10 minutes).
Rinse briefly with 1% aqueous acetic acid.
Dehydrate rapidly with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – red
Fresh fibrin – yellow
Erythrocytes – yellow
Connective tissue – blue

Notes

Crystal scarlet is more commonly known as ponceau 6R.
Steps 10 & 11 specify the time as up to. These times should be established by inspection, but will generally remain consistent.
Lendrum’s recommendation for formalin fixed material was to dewax sections and treat with trichlorethylene in a sealed contained for 48 hours at 56°C, then to refix in absolute ethanol saturated with both picric acid and mercuric chloride for 24 hours before proceeding to step 2. The alternative treatment with Bouin’s fluid given at step 3 is often satisfactory.
Bancroft notes that dyes other than those originally given may be used. Some may not be easily available, and no CI numbers were given.
Color Dye Options
Yellow Lissamine fast yellow
Blue Durazol blue
Pontamine sky blue
Fast green FCF
Naphthalene black 10B
Red Ponceau de xylidine
Azofuchsin

Color	Dye Options
Yellow	Lissamine fast yellow
Blue	Durazol blue Pontamine sky blue Fast green FCF Naphthalene black 10B
Red	Ponceau de xylidine Azofuchsin

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Obadiah for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Obadiah

for Fibrin

steps

materials

print

The name of this stain comes from the letters OBDR45, which refer to the dyes used: Orange, Blue, Direct Red 45 (a synonym for one of the red dyes).

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Results are usually poor with formalin fixed material, even if treated with Bouin’s fluid at 56°C for an hour or so.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Picro-mercuric ethanol
Material Amount
Ethanol, absolute 100 mL
Picric acid to saturation
Mercuric chloride to saturation
Orange
Material Amount
Orange G 0.5 g
Phosphotungstic acid 1 g
Ethanol, 95% 100 mL
Blue
Material Amount
Naphthalene blue black CS 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Red – Option 1
Material Amount
Chicago red 2.5 g
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Red – Option 2
Material Amount
Polar brilliant red BN 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Ethanol, absolute	100	mL
Picric acid	to	saturation
Mercuric chloride	to	saturation

Material	Amount
Orange G	0.5	g
Phosphotungstic acid	1	g
Ethanol, 95%	100	mL

Material	Amount
Naphthalene blue black CS	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Chicago red	2.5	g
Acetic acid, glacial	2.5	mL
Distilled water	97.5	mL

Material	Amount
Polar brilliant red BN	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, as this produces tissues that stain poorly. Sections should be 3-5 µ thick.

Protocol

Dewax sections with xylene.
Place into a sealed container of trichlorethylene at 56°C for 48 hours.
Rinse well with absolute ethanol.
Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
Remove mercury pigment with the iodine-thiosulphate sequence.
Stain nuclei with an acid resistant nuclear stain.
Place in the orange stain for 2 minutes.
Rinse with distilled water.
Place in the Blue stain up to 30 minutes.
Differentiate with the polyacid for 5 minutes.
Place in the Red stain 15-20 minutes (polar brilliant red) or 15-30 minutes (chicago red).
Rinse with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Old fibrin – black
Younger fibrin – may be yellow
Connective tissue – red

Notes

Note that this method reverses the usual order and uses a blue stain before the red stain.
Lendrum considered that this method demonstrated fibrin that other methods stained as collagen.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Picro-Mallory for Fibrin – Long Version

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Picro-Mallory

for Fibrin – Long Version

steps

materials

print

Lendrum, Fraser and others published several fibrin stains, the most well known being the Picro-Mallory. There are several variants of the method, some more complicated than others.

See this alternative protocol for a shorter version.

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Adequate results can be obtained with thorough formalin fixation (24 hours), thorough processing (overnight), sectioning, followed by refixing in Bouin’s fluid at 56°C for an hour or so. However, results are inferior to those obtained by the full procedure. It is also a method where experience is required for optimal results.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the
celestine blue-hemalum sequence.
Yellow mordant
Material Amount
Picric acid, sat. in 80% ethanol 100 mL
Orange G 0.2 g
Lissamine yellow 0.2 g
Stock differentiator
Material Amount
Picric acid 2.5 g
Ethanol, 95% 100 mL
Phosphotungstic acid 25 g
Dissolve ingredients and filter.
Red differentiator
Material Amount
Stock differentiator 40 mL
Ethanol, 95% 20 mL
Distilled water 90 mL
Blue differentiator
Material Amount
Stock differentiator 10 mL
Distilled water 90 mL
Red stain
Material Option 1 Option 2 Option 3
Acid fuchsin 1 g – 0.4 g
Lissamine fast red – – 0.2 g
Biebrich scarlet – 1 g –
Acetic acid, glacial 1 mL 1 mL 1 mL
Distilled water 99 mL 99 mL 99 mL
Blue stain
Material Amount
Methyl blue 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Picric acid, sat. in 80% ethanol	100	mL
Orange G	0.2	g
Lissamine yellow	0.2	g

Material	Amount
Picric acid	2.5	g
Ethanol, 95%	100	mL
Phosphotungstic acid	25	g

Material	Amount
Stock differentiator	40	mL
Ethanol, 95%	20	mL
Distilled water	90	mL

Material	Amount
Stock differentiator	10	mL
Distilled water	90	mL

Material	Option 1	Option 2	Option 3
Acid fuchsin	1	g	–	0.4	g
Lissamine fast red	–	–	0.2	g
Biebrich scarlet	–	1	g	–
Acetic acid, glacial	1	mL	1	mL	1	mL
Distilled water	99	mL	99	mL	99	mL

Material	Amount
Methyl blue	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, especially with formalin fixatives, as this produces tissues that stain poorly even with secondary fixation such as Bouin’s fluid at 56°C for an hour. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment with the iodine-thiosulphate sequence.
Stain nuclei with an acid resistant nuclear stain.
Place in yellow mordant for 2-3 minutes.
Wash in distilled water until only erythrocytes are yellow.
Place in the red stain for 5-10 minutes.
Rinse with 1% aqueous acetic acid.
Differentiate with the red differentiator until fibrin is prominent microscopically.
Rinse well in distilled water.
Place in the blue stain for 5 minutes.
Rinse briefly with 1% aqueous acetic acid.
Place in the blue differentiator for 1-2 minutes.
Biefly rinse with 1% aqueous acetic acid.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – clear red
Erythrocytes – yellow
Connective tissue – blue

Notes

The most common dye combination is acid fuchsin and methyl blue.
Adequate results may be obtained with 24-48 hours fixation in neutral buffered formalin, overnight paraffin processing and secondary fixation of sections with Bouin’s picro-formol-acetic for 1 hour at 56°C.
Good results are obtained with the fixation and processing recommended in the text.
Optimal results are obtained with the fixation and processing recommended in the text, followed by degreasing and secondary fixation in picro-mercuric ethanol.
Replace step 1 with the following:
1. Dewax sections with xylene.
2. Place in trichlorethylene in a sealed container at 56°C for 48 hours.
3. Rinse sections well with absolute ethanol.
4. Place in picro-mercuric-ethanol for 24 hours.
5. Wash sections with water, and continue from step 2.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Picro-Mallory for Fibrin – Shorter Version

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Picro-Mallory

for Fibrin – Shorter Version

steps

materials

print

This version of the picro-Mallory is relatively uncomplicated and is suitable for a routine pathology laboratory. Proper fixation is still essential and minimalist formalin fixation with quick processing should be avoided as it will give disappointing results with poorly stained erythrocytes. Originally, extended mercury fixation, thorough processing, degreasing and refixing in picro-mercuric-ethanol were specified. Although fixation in formol sublimate (or B5) is preferred for this method as well, adequate results can be obtained with overnight formalin fixation and overnight processing. Refixation of sections in Bouin’s fluid at 56°C for an hour or so overcomes some of the deficiencies in fixation, but results are still inferior to those obtained by the full procedure.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Picro-orange
Material Amount
Picric acid, sat. in 80% ethanol 100 mL
Orange G 0.2 g
Yellow differentiator
Material Amount
Picro-orange 30 mL
Ethanol, 80% 70 mL
Red stain
Material Amount
Acid fuchsin 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Red differentiator
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL
Blue stain
Material Amount
Methyl blue 2 g
Acetic acid, glacial 2 mL
Distilled water 98 mL

Material	Amount
Picric acid, sat. in 80% ethanol	100	mL
Orange G	0.2	g

Material	Amount
Picro-orange	30	mL
Ethanol, 80%	70	mL

Material	Amount
Acid fuchsin	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Material	Amount
Methyl blue	2	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment with the iodine-thiosulphate sequence.
Place into Bouin’s fluid at 56°C for 1 hour.
Rinse well in distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in picro-orange for 2 minutes.
Without rinsing, place in the red stain for 5 minutes.
Rinse with distilled water.
Dip into yellow differentiator.
Rinse in distilled water.
Differentiate with the red differentiator for 5 minutes.
Rinse with distilled water.
Place in the blue stain for 2 minutes.
Rinse briefly with 1% aqueous acetic acid.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – clear red
Fibrinoid – varies from red to orange
Erythrocytes – orange
Connective tissue – blue

Notes

If possible, the extended fixation and slow processing specified for the longer version should be used. Degreasing and refixation will also improve results if time allows.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.

March 30, 2023

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Yellowsolve I for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Yellowsolve I

for Fibrin

steps

materials

print

Materials

Celestine blue-hemalum nuclear stain, or similar.
Trichlorethylene
Tetrachlorethylene
Solution A
Material Amount
Phloxine B 0.5 g
Calcium chloride 0.5 g
Distilled water 100 mL
Solution B
Material Amount
Lissamine flavine FF to saturation
2-Ethoxyethanol 50 mL
Ethyl phosphate 50 mL
Optional – an elastic fibre stain: Orcein or Weigert’s iron resorcin fuchsin, or variant.

Material	Amount
Phloxine B	0.5	g
Calcium chloride	0.5	g
Distilled water	100	mL

Material	Amount
Lissamine flavine FF	to saturation
2-Ethoxyethanol	50	mL
Ethyl phosphate	50	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Protocol

Dewax sections with xylene.
Place in trichlorethylene for 24 to 48 hours.
Bring sections to water.
Stain elastic fibres if desired.
Stain nuclei with the celestine blue-hemalum sequence.
Wash well with water.
Place in solution A for 30 minutes.
Rinse with 2-ethoxyethanol then with tetrachlorethylene.
Place into tetrachlorethylene overnight.
Rinse with 2-ethoxyethanol.
Differentiate with solution B, controlling microscopically.
Rinse with 2-ethoxyethanol.
Clear in xylene and mount with a synthetic resinous medium.

Expected Results

Fibrin – red
Background – yellow
Nuclei – black
Acidophil cytoplasmic inclusions – red

Notes

2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH₃CH₂OCH₂CH₂OH.
Tetrachlorethylene has the formula Cl₂C=CCl₂ or C₂Cl₄
Trichlorethylene has the formula ClHC=CCl₂ or C₂HCl₃
Both trichlorethylene and tetrachlorethylene should be used in a fume hood.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Garvey’s Stain for Elastic, Fibrin & Collagen

By Carmen WongDye Type, Elastic Fibers, Fibrin, Iron Hematoxylin, Protocols, Stain Target, Stain Type

Protocol

Garvey's Stain

for Elastic, Fibrin & Collagen

steps

materials

print

Materials

Stock Verhoeff A

Material	Amount
Hematoxylin	3	g
Ethanol, absolute	100	mL

Stock Verhoeff B

Material	Amount
Ferric chloride	2.3	g
Distilled water	100	mL

Stock Verhoeff C

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working Verhoeff

Material	Amount
Stock solution A	3	volumes
Stock solution B	2	volumes
Stock solution C	1	volume

Polyacid

Material	Amount
Phosphotungstic acid	2	g
Distilled water	100	mL

Acetic water

Material	Amount
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Picro-mercuric chloride

Material	Amount
Picric acid, sat. aqu.	100	mL
Mercuric chloride	5	g
Distilled water	100	mL

Erythrocyte stain

Material	Amount
Lissamine fast yellow	1	g
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Plasma stain

Material	Amount
Biebrich scarlet	0.75	g
Acid fuchsin	0.75	g
Ponceau 2R	0.75	g
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Material	Amount for Var I		Amount for Var II
Aniline blue	0.5	g	–
Light green SF yellowish	–		2	g
Acetic acid, glacial	1	mL	2	mL
Distilled water	100	mL	100	mL

Tissue Sample

Formol sublimate fixation is preferred, although 10% formalin variants are acceptable. Paraffin sections at 3µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
1. Optionally, place into picro-mercuric chloride for 1 hour.
2. Wash well with warm water to remove picric acid.
Place in verhoeff’s solution for 9 minutes.
Wash with warm tap water for 5 minutes.
Place in lissamine fast yellow for 2 minutes.
Rinse with 0.5% acetic acid.
Place in the plasma stain for 5 minutes.
Rinse with distilled water.
Place in polyacid for 10 minutes.
Rinse with distilled water.
Place in a fiber stain variant for 2 minutes.
Rinse with 0.5% acetic acid.
Dehydrate, clear and mount with a resinous medium.

Expected Results

Nuclei – black
Elastic fibres – black
Fibrin – young – yellow
Fibrin – mature – red
Fibrin – old – blue or green
Erythrocytes – yellow
Muscle – red
Collagen – blue or green (some may be red)

Notes

Sections should be treated with picro-mercuric chloride if formol sublimate or similar fixation was not used initially.
The ferric chloride should be the hexahydrate crystals.
Although the iodine content of the Verhoeff elastic stain component may remove any mercury pigment, in methods such as this, some technologists prefer to apply the iodine-thiosulphate sequence before staining.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Garvey W., et. al., (1987),
A combined elastic, fibrin and collagen stain
Stain Technology, V. 62, No 6, pp 365-367.

March 30, 2023

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Gram Weigert for Fibrin and Gram Positive Bacteria

By Carmen WongBacteria, Fibrin, Gram Staining, Protocols, Stain Target, Stain Type

Protocol

Gram Weigert

for Fibrin and Gram Positive Bacteria

steps

materials

print

Materials

Eosin

Material	Amount
Eosin Y ws	5	g
Distilled water	100	mL

Crystal violet

Material	Amount
Crystal violet	1	g
Distilled water	100	mL

Gram’s iodine

Material	Amount
Iodine	2	g
Potassium iodide	4	g
Distilled water	400	mL

Aniline-Xylene

Material	Amount
Aniline	1	volume
Xylene	1	volume

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place in eosin solution for 5 minutes.
Rinse with tap water.
Place in crystal violet 1 minute.
Rinse with tap water.
Flood with Gram’s iodine for 1 minute.
Rinse with tap water.
Gently blot the section, being careful not to damage it.
Decolorise the section with aniline-xylene.
Rinse with several changes of xylene to remove all aniline.
Mount with a resinous medium.

Expected Results

Gram positive bacteria – blue
Fibrin – blue
Background – pink

Notes

Control the differentiation with aniline-xylene microscopically. To examine, place the section in xylene to stop dye removal. Return to aniline-xylene if more differentiation is needed. Stop differentiation when the target element has good contrast.
Increasing the aniline content of the aniline-xylene will increase the speed of dye removal. Decreasing it will slow dye removal.
If the background is not pink enough, increase the time in eosin, stain in eosin at elevated temperature, or increase the eosin concentration.
The eosin counterstain may be omitted entirely if wished.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Culling, C.F.A., (1963)
Handbook of histopathological techniques, 2nd ed.
Butterworths, London.
McManus, J.F.A. and Mowry, R.W., (1960),
Staining methods, histologic and histochemical,
Harper & Row, New York, NY, USA.

March 30, 2023

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Masson 44/41 for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Masson 44/41

for Fibrin

steps

materials

print

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Picro-mercuric ethanol
Material Amount
Ethanol, absolute 100 mL
Picric acid to saturation
Mercuric chloride to saturation
Plasma stain
Material Amount
Ponceau 6R 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Fibre stain
Material Amount
Naphthalene blue black CS 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Polyacid
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL

Material	Amount
Ethanol, absolute	100	mL
Picric acid	to saturation
Mercuric chloride	to saturation

Material	Amount
Ponceau 6R	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Naphthalene blue black CS	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Tissue Sample

Protocol

Dewax sections with xylene.
Place into a sealed container of trichlorethylene at 56°C for 48 hours.
Rinse well with absolute ethanol.
Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
Remove mercury pigment with the iodine-thiosulphate sequence.
Stain nuclei with an acid resistant nuclear stain.
Place in the plasma stain for 5 minutes.
Differentiate with the polyacid for 5 minutes.
Place in the fibre stain for 30 minutes.
Rinse briefly with 1% aqueous acetic acid.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fresh fibrin – red
Older fibrin – black
Connective tissue – pale blue
Plasma cell inclusions – red

Notes

Ponceau 6R is also known as acid red 44.
Naphthalene blue black CS is also known as acid black 41.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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