Skip to main content
search
Category

Trichrome, One-Step

Wallart & Honette’s Trichrome for Connective Tissues

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Wallart & Honette's Trichrome

for Connective Tissues

9
steps
6
materials
print

Materials

Stock solution 1

MaterialAmount
Acid fuchsin1g
Acetic acid1mL
Distilled water100mL

Stock solution 2

MaterialAmount
Fast yellow3g
Acetic acid1mL
Distilled water100mL

Stock solution 3

MaterialAmount
Phosphomolybdic acid1g
Distilled water100mL

Working solution A

MaterialAmount
Stock solution 130mL
Stock solution 230mL
Stock solution 330mL

Working solution B

MaterialAmount
Acetic acid1mL
Distilled water100mL

Working solution C

MaterialAmount
Acetic acid1mL
Ethanol 100%100mL

Tissue Sample

5µ paraffin sections of formalin fixed tissue are suitable. Many other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with an acid resistant nuclear stain.
  3. Rinse well with tap water.
  4. Place into solution A for 5 minute.
  5. Rinse quickly with distilled water.
  6. Place into solution B for 5 minutes.
  7. With a pipette, drop solution C onto the slide for 30 seconds.
  8. Dehydrate with ethanol.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic  –  pink
  • Cytoplasm  –  red
  • Collagen  –  yellow
  • Nuclei  –  black

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Wallart and Honette, (1934)
    Bulletin d’histologie appliquée à la physiologie et à la pathologie et de technique microscopique, v. 10, p. 404.
March 30, 2023
Love0

Walter’s Trichrome for Elastic and Collagen

By Dye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Walter's Trichrome

for Elastic and Collagen

9
steps
8
materials
print

Materials

Solution A

MaterialAmount
Ferric ammonium sulphate2.5g
Distilled water100mL

Solution B

MaterialAmount
Phosphotungstic acid2g
Distilled water100mL

Solution C

MaterialAmount
Eosin B0.7g
Acid fuchsin, sat. aqu4mL
Unna’s elastic stain35mL
Ethanol, 50%35mL
Glycerol40mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into solution A for 24 hours.
  3. Rinse quickly with distilled water.
  4. Place into solution B for 10 minutes.
  5. Rinse quickly with distilled water.
  6. Place into solution C for 15-20 minutes.
  7. Rinse quickly with 95% ethanol until clouds of dye stop being extracted.
  8. Dehydrate with 100% ethanol for 1 minute.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  purple
  • Elastic fibres  –  purple
  • Erythrocytes  –  orange
  • Collagen  –  blue

Notes

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Walter, (1930)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskipische Technik,
    v. 46, pp. 458
March 30, 2023
Love0

Pollak’s Trichrome for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Pollak's Trichrome

for Muscle and Collagen

6
steps
11
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialAmount
    Orange G0.25g
    Ponceau 2R0.33g
    Acid fuchsin0.17g
    Light green SF0.15g
    Phosphotungstic acid0.5g
    Phosphomolybdic acid0.5g
    Acetic acid, glacial1mL
    Ethanol, 95%50mL
    Distilled water50mL
  • Solution B
    MaterialAmount
    Acetic acid, glacial0.2mL
    Distilled water100mL

Preparation of Solution A

  1. Mix the water, ethanol and acetic acid together.
  2. Divide into four, and dissolve as follows:
    1. Phosphomolybdic acid
    2. Phosphotungstic acid and orange G
    3. Light green SF
    4. Ponceau 2R and acid fuchsin
  3. Combine all solutions and filter.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert’s iron hematoxylin or equivalent.
  3. Place into solution A for 3-7 minute.
  4. Place into solution B until differentiated.
  5. Dehydrate with ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Cytoplasm  –  red
  • Muscle  –  red
  • Collagen  –  blue

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Pollack, (1944)
    Archives of pathology and laboratory medicine, v. 37, pp. 294
    Chicago, USA
March 30, 2023
Love0

Sweat, Meloan & Puchtler’s One Step Trichrome

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Sweat, Meloan & Puchtler's

One Step Trichrome

7
steps
6
materials
print

Materials

Solution A

MaterialAmount
Chromotrope 2R0.6g
Aniline blue0.6g
Phosphomolybdic acid1g
Hydrochloric acid, conc.1mL
Distilled water100mL

Solution B

MaterialAmount
Acetic acid, glacial1mL
Distilled water100mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in Bouin’s fixative at 56°C for one hour.
  3. Wash with running tap water 3 to 4 minutes to remove yellow.
  4. Place into solution A for 1 minute.
  5. Rinse with solution B for 30 seconds.
  6. Dehydrate rapidly with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei, cytoplasm, muscle, fibrin & elastic  –  red
  • Collagen  –  blue

Notes

  • A nuclear stain is not used as the hydrochloric acid will remove it.

Safety Note

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

References

  1. Culling, C.F.A., Alison, R.T. and Barr, W.T. (1985)
    Cellular Pathology Technique, 4th ed.
    Butterworths, London, UK.
March 30, 2023
Love0

McFarlane’s Trichrome 1st Variant for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

McFarlane's Trichrome 1st Variant

for Muscle and Collagen

10
steps
9
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialAmount
    Picric acid0.2g
    Acid fuchsin1g
    Aniline blue2g
    Phosphotungstic acid1g
    Acetic acid, glacial2mL
    Distilled water98mL
  • Solution B
    MaterialAmount
    Acetic acid, glacial2mL
    Distilled water100mL
  • Solution C
    MaterialAmount
    Picric acid0.25g
    Phosphotungstic acid2.5g
    Ethanol, 95%10mL
    Distilled water90mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert’s iron hematoxylin or equivalent.
  3. Place into solution A for 5 minute.
  4. Rinse with solution B.
  5. Rinse with distilled water.
  6. Place into solution C until differentiated.
  7. Rinse with distilled water.
  8. Wash with solution B.
  9. Dehydrate with ethanol.
  10. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Muscle  –  red
  • Cytoplasm  –  red
  • Collagen  –  blue

Notes

  • McFarlane also published two other, multi-step, trichrome methods. One is simpler than the other.
  • Solutions A and C require dry weights of picric acid. This could be provided from a saturated aqueous solution (1 g in 78 mL). Alternate formulas are:

    Solution A

    MaterialAmount
    Picric acid, sat. aqueous16mL
    Distilled water82mL
    Acetic acid, glacial2mL
    Acid fuchsin1g
    Aniline blue2g
    Phosphotungstic acid1g

    Solution C

    MaterialAmount
    Picric acid, sat. aqueous20mL
    Distilled water70mL
    Ethanol, 95%10mL
    Phosphotungstic acid2.5g

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    McFarlane, (1944)
    Stain Technology, v. 19, pp. 29
March 30, 2023
Love0

Kostowiecki’s Trichrome for Muscle, Cartilage and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Kostowiecki's Trichrome

for Muscle, Cartilage and Collagen

7
steps
5
materials
print

Materials

  • A red nuclear stain
  • Solution A
    MaterialAmount
    Aniline blue0.06g
    Orange G0.2g
    Phosphomolybdic acid1g
    Distilled water100mL

Preparation of Solution A

  1. Add both dyes to the water.
  2. Bring to the boil for three minutes.
  3. Add the phosphomolybdic acid while hot.
  4. Cool and filter.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with a red nuclear stain.
  3. Place into the staining solution until darkly stained, 30-120 minutes.
  4. Rinse with distilled water.
  5. Place in 95% ethanol for 1 minute.
  6. Dehydrate with absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Muscle  –  orange
  • Cartilage  –  blue
  • Collagen  –  blue-green

Safety Note

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


References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Kostowiecki, (1932)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v. 49, pp. 337
March 30, 2023
Love0

Engel & Cunningham’s Trichrome for Muscle Fibres, Types I & II

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Engel & Cunningham's Trichrome

for Muscle Fibres, Types I & II

7
steps
6
materials
print

Materials

  • Harris alum hematoxylin
  • Solution A
    MaterialAmount
    Chromotrope 2R0.6g
    Fast green FCF0.3g
    Phosphotungstic acid0.6g
    Acetic acid, glacial1mL
    Distilled water99mL

    Adjust to pH 3.4 with 1N NaOH.

  • Solution B
    MaterialAmount
    Acetic acid, glacial0.2mL
    Distilled water100mL

Tissue Sample

Unfixed frozen sections of liquid nitrogen quenched muscle. Sections should be 5-10µ in thickness. The fibres should be in cross section, as close to a right angle to their length as possible.

Protocol

  1. Cut cryostat sections and dry at room temperature.
  2. Stain nuclei with Harris’ hematoxylin for 5 minutes.
  3. Rinse well with distilled water.
  4. Place into solution A for 10 minute.
  5. Rinse with solution B for a few seconds to differentiate.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Type I fibres  –  green with a red cast
  • Type II fibres  –  green

Notes

  • Both fibre types are green overall, but type I fibres contain more red staining granular material and have a red cast.
  • This is a modification of Gomori’s trichrome.

Safety Note

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

References

  1. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques, Ed. 3
    Butterworth, London, UK.
    Citing:
    Engel & Cunningham, (1963)
    Rapid examination of muscle tissue.
    An improved trichrome method for fresh frozen biopsy specimens.
    Neurology, vol.13, pp.921
March 30, 2023
Love0

Papanicolaou’s Trichrome for Exfoliated Cells

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Papanicolaou's Trichrome

for Exfoliated Cells

7
steps
8
materials
print

This is not the Papanicolaou technique commonly used to screen for cervical cancer.

Materials

Tissue Sample

Ethanol fixed smears of exfoliated epithelial cells.

Protocol

  1. Stain nuclei with Ehrlich’s hematoxylin for 2 minutes.
  2. Blue.
  3. Rinse with distilled water.
  4. Place into the staining solution for 2-5 minutes.
  5. Rinse quickly with distilled water.
  6. Dehydrate rapidly with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cells  –  shades of orange, pink or blue

Notes

  • The method specified that dehydration and clearing should be with dioxane. This reagent is no longer used due to its toxicity. If satisfactory results are not obtained with rapid ethanol dehydration, dehydrants such as t-butanol should be tried.

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Papanicolaou, (1941)
    Journal of laboratory and clinical medicine, v. 26, pp. 1200
March 30, 2023
Love0

Pasini’s Trichrome for Elastic and Collagen

By Dye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Pasini's Trichrome

for Elastic and Collagen

8
steps
8
materials
print

Materials

  • Ehrlich’s alum hematoxylin
  • Solution A
    MaterialAmount
    Phosphotungstic acid2g
    Distilled water100mL
  • Solution B – Var I
    MaterialAmount
    Eosin B0.7g
    Acid fuchsin, sat. aqu.4mL
    Unna’s elastic stain35mL
    Glycerol40mL
    Ethanol, 50%35mL
  • Solution B – Var II
    MaterialAmount
    Eosin B, 2% in 50% ethanol30mL
    Acid fuchsin, sat. aqu.4mL
    Unna’s elastic stain30mL
    Glycerol15mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with an alum hematoxylin nuclear stain.
  3. Place into solution A for 10 minute.
  4. Rinse quickly with distilled water.
  5. Place into solution B for 15-20 minutes.
  6. Rinse quickly with 70% ethanol.
  7. Dehydrate and differentiate with 100% ethanol for a few seconds.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Elastic fibres  –  purple
  • Erythrocytes  –  orange
  • Collagen  –  blue

Notes

  • Solution B var I was given by Gray, var II was given by Gatenby & Beams.

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Gatenby, J. B. & Cowdry, E. V., (1928)
    Bolles Lee’s Microtomist’s Vade Mecum, pp. 280
    Blakiston, Philadelphia
  2. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed., pp. 485
    Churchill, London, UK.
March 30, 2023
Love0

Gomori’s Trichrome for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Gomori's Trichrome

for Muscle and Collagen

7
steps
6
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialAmount
    Chromotrope 2R0.6g
    Fast green FCF0.3g
    Phosphotungstic acid0.6g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Solution B
    MaterialAmount
    Acetic acid, glacial0.2mL
    Distilled water100mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with an acid resistant nuclear stain.
  3. Place into solution A for 5-20 minute.
  4. Quickly rinse off stain with distilled water.
  5. Rinse with solution B.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Cytoplasm  –  red
  • Collagen  –  green

Notes

  • The time in solution A should be determined empirically. Once a satisfactory stain has been achieved, the time should remain constant. Any change in fixation, processing or section thickness warrants reviewing the staining time.
  • Light green SF yellowish can replace Fast green FCF.
  • Wheatley recommended this trichrome for protozoa in intestinal smears.
  • Engel & Cunningham modified this method to differentiate muscle fibre types in cryostat sections.

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Gomori, (1950),
    Technical Bulletin, vol.20, pp.77
    And:
    Wheatley, (1951)
    Technical Bulletin, vol.21, pp.92
  2. Humason, G. L., (1967)
    Animal tissue techniques, Ed. 2
    W. H. Freeman and Company, San Francisco, Ca, USA
March 30, 2023
Love0