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Mordanted Hematoxylin

Mallory’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mallory's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematoxylin2.5 gDye
Potassium alum50 gMordant
Distilled water1 LSolvent
Thymol2.5 gPreservative

Compounding Procedure

  1. Dissolve the dye and Alum in the water.
  2. Add the thymol.
  3. Allow to ripen before use.

    4. </ol

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
March 30, 2023
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Mallory & Wright’s Alum Hematoxylin Variants

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mallory & Wright's Alum Hematoxylin Variants

8
steps
3
materials
print

Materials

MaterialStandard FormulaStrong FormulaFunction
Hematoxylin1 g1 gDye
Ammonium alum, saturated aqueous100 mL100 mLMordant
Distilled water300 mL–Solvent

Compounding Procedure

  1. Dissolve the hematoxylin in the Alum solution by warming if necessary.
  2. Add the water if the standard solution is being used.
  3. Plug the container with cotton wool.
  4. Ripen for approximately ten days.
  5. Place the stopper tightly.
  6. Filter before use. The solution is stable for 2-3 months.
  7. A small crystal of thymol may be added.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The standard solution is for routine, formalin fixed tissues.
  • The strong solution is recommended for Zenker fixed tissues.
  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

  1. Mallory, F. B. & Wright, J.H., (1904)
    Pathological technique, Ed.3
    W. B. Saunders, Philadelphia, USA.
March 30, 2023
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Mallory’s PTAH

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mallory's PTAH

6
steps
3
materials
print

Materials

MaterialAmountFunction
Hematein,1 g.Dye
Phosphotungstic acid10 gMordant
Distilled water1 LSolvent

Compounding Procedure

  1. Dissolve the hematein in 200 mL of the water and the phosphotungstic acid in the rest.
  2. Combine the solutions.
  3. Keep in a bottle with a tightly fitting cap to ensure atmospheric oxygen is excluded.
  4. The stain may be used after a day or two.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Perform a Mallory bleach using 0.25% potassium permanganate.
  3. Rinse well with water.
  4. Place into the PTAH solution for 12 – 24 hours at room temperature.
  5. Rinse well with water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei, erythrocytes, fresh fibrin, muscle striations  –  blue
  • Background  –  red

Notes

  • If formalin fixed, the sections may be treated with acid dichromate for 30 minutes.
    MaterialAmount
    Potassium dichromate, 3% aqueous36mL
    10% hydrochloric acid in absolute ethanol.12mL
  • The staining may be done in an oven at about 60°C for a few hours, but the results are often less satisfactory.
  • Many substances have been stated to be stained blue. Sometimes the red staining overshadows blue staining, and the section may need to be washed with water to remove excess red. Excess blue may be removed by extending the treatment with ethanol during dehydration.
  • PTAH may also be made with hematoxylin and oxidized as any other hematoxylin solution. Atmospheric oxidation is often recommended, but takes a few months. Place the solution in a flask with a loose cotton batting stopper to facilitate exposure to air. Test periodically and, when staining is satisfactory, place into a bottle with a tightly fitting lid to inhibit atmospheric oxidation.
  • Alternatively, the hematoxylin may be chemically oxidized. This is usually done with 12 mL of 1% aqueous potassium permanganate. The solution may be used after a day or two. Sodium iodate may also be used. Half the hematoxylin will be oxidized with 0.1 grams sodium iodate. If brought to a boil and allowed to cool, it may be used immediately, or it may be allowed to oxidize at room temperature for a few days.

Safety Note

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

References

  1. Bancroft, J. D. and Stevens, A.,
    Theory and Practice of Histological Techniques, Ed. 2,
    Churchill Livingstone, London, UK.
March 30, 2023
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Mann’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mann's Alum Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematein6 gDye
Potassium alum35 gMordant
Distilled water350 mLSolvent
95% ethanol320 mLSolvent
Glycerol250 mLStabilizer
Glacial acetic acid30 mLAcidifier

Compounding Procedure

  1. Dissolve the dye in the acetic acid.
  2. Mix the ethanol and glycerol together, and add to the dye in acetic acid.
  3. Dissolve the Alum in the water, and add to the dye solution.
  4. The solution may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • This solution uses hematein instead of hematoxylin.
  • The high dye content indicates this is a strong, regressive solution.
  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
March 30, 2023
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Martinotti’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Martinotti's Alum Hematoxylin

8
steps
5
materials
print

Materials

MaterialAmountFunction
Hematein2 gDye
Ammonium alum15 gMordant
Distilled water700 mLSolvent
Methanol150 mLSolvent
Glycerol150 mLStabiliser

Compounding Procedure

  1. Dissolve the Alum in 600 mL water.
  2. Dissolve the hematein in 100 mL water.
  3. Combine, and add the other ingredients.
  4. The solution may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • This solution uses hematein instead of hematoxylin.
  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
March 30, 2023
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Masson’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Masson's Iron Hematoxylin

8
steps
3
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate4 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Hematoxylin1 gDye
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solution B should be ripened for a minimum of one month.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 5-10 minutes preheated to 50°C.
  3. Rinse with distilled water.
  4. Place into solution B for 5-10 minutes preheated to 50°C.
  5. Rinse with tap water.
  6. Differentiate in solution A at room temperature, controlling microscopically.
  7. Wash well in running tap water to blue.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei and other structures  –  blue to black

Notes

  • This is a modification of Heidenhain’s iron hematoxylin, and differs solely in the concentration of the reagents, and the temperature at which staining is carried out.
  • The stock solutions are stable for some time.
  • The hematoxylin solution needs to be ripened.
  • The degree of differentiation will determine which tissue components are prominent. The method can demonstrate many structures, including chromosomes, nuclear components, mitochondria and muscle striations
  • The solutions may be reused, with the exception of the solution A used to differentiate, which should be fresh each time.
  • Counterstaining is not recommended.
  • This method is usually recommended for monochrome photography.

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:
    Heidenhain, M., (1892).
    Festschrift Herrn A. von Kolloker zur Feier seines fünfzigjährigen medicinischen
    Doktorjubiläums, p.118. Wilhelm Engellmans, Leipzig, Germany
    and:
    Masson, M.,, (1892).
    Bulletin et mémoires de la Société anatomique de Paris, v.87. p.291.
March 30, 2023
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Mitchell’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mitchell's Alum Hematoxylin

6
steps
4
materials
print

Mitchell’s formula is from 1883 and is now obsolete. It does, however, show how Alum hematoxylin solutions were originally prepared, and the variability inherent in the procedures for doing so. The modern formula should stain satisfactorily.

Materials

Original Formula

MaterialAmountFunction
Logwood, ground2 ouncesDye
Potassium alum9 ouncesMordant
Distilled wateras neededSolvent
Glycerol4 fl. ouncesStabiliser

Compounding Procedures

  1. Moisten the ground logwood with water and pack it into a funnel.
  2. Pour water onto the wood until it comes through barely coloured.
  3. Remove the wood from the funnel, spread out and dry.
  4. Dissolve the Alum in 8 fluid oz of water.
  5. Moisten the logwood with some Alum water and pack it tightly into the funnel.
  6. Pour the rest of the Alum solution onto the logwood.
  7. When the first drops come through, seal up the tip of the funnel.
  8. Leave 48 hours for the dye to be extracted.
  9. Remove the seal and collect the fluid that comes through.
  10. Pour on more water until 12 fluid oz have been collected.
  11. Add the glycerol, mix well and filter.

Modern Formula

MaterialAmountFunction
Hematoxylin3 gDye
Potassium alum35 gMordant
Distilled water340 mLSolvent
Glycerol115 mLStabiliser

Compounding Procedures

  1. Dissolve the Alum in the water.
  2. Add the hematoxylin and mix until dissolved.
  3. Add the glycerol, mix well and filter.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for a few minutes, or dilute 1:7 with distilled water and stain overnight.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The staining characteristics were not given, but it is likely progressive, especially when diluted. If overstaining does occur, differentiate with 0.5 – 1% hydrochloric acid in 70% ethanol.
  • The reason for the pre-wash of the logwood in water during the original preparation method was stated to be for the removal of tannin.
  • The amount of hematoxylin in the modern formula is based on the fact that logwood contains up to 10% of the dye. Two ounces (about 57g) could contain a maximum of about 6 grams of dye. A 50% extraction, or 3 grams hematoxylin has been allowed for. It could well have been more or less than this.
  • The original formula gives 255 grams of potassium Alum to dissolve in 227 mL water. It takes just over 7 mL water to dissolve 1 gram crystalline potassium alum, so the amount of water specified would be able to dissolve only about 32 grams.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

  1. Arthur Bolles-Lee, (1885)
    The Microtomist’s Vade-Mecum
    Originally published by: J & A Churchill, London, England.
    Republished by: Science Heritage Ltd., Lincolnwood, Illinois, USA.
  2. Susan Budavari, Editor, (1996)
    The Merck Index, Ed. 12
    Merck & Co., Inc., Whitehouse Station, NJ, USA
  3. Lemmens, R. H. M. J. and Wulijarna-Soetjipto, N., Editors. (1992)
    Plant resources of South East Asia No. 3,
    Dye and tannin-producing plants.
    PROSEA, Bogor, Indonesia.
March 30, 2023
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Morel & Bassal’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Morel & Bassal's Iron Hematoxylin

8
steps
6
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin1 gDye
95% ethanolmLSolvent

Solution B

MaterialAmountFunction
Ferric chloride2 gMordant
Cupric acetate0.04 gMordant
Distilled water100 mLSolvent
Hydrochloric acid1 mLAcidifier

Compounding Procedure

  1. Make each solution separately.
  2. For use, combine equal parts of solutions A and B.
  3. The working solution may be used immediately, but is not stable for long.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with tap water.
  4. Dip briefly in 1% hydrochloric acid in 70% ethanol.
  5. Wash well in running tap water to blue.
  6. Rinse with distilled water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The stock solutions are stable for some time.
  • The working solution should be made fresh, and is likely not stable for long.
  • The staining time should be determined by trial.

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:
    Morel and Bassal, (1909)
    Journal de l’anatomie et de la physiologie normales et pathologique de l’homme
    et des animaux, v. 45, p. 632.
March 30, 2023
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Murray’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Murray's Iron Hematoxylin

8
steps
4
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate3.5 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solution B should be ripened for a minimum of one month.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 30 minutes to 24 hours.
  3. Rinse with distilled water.
  4. Place into solution B for 30 minutes to 24 hours.
  5. Rinse with tap water.
  6. Differentiate in solution A, controlling microscopically.
  7. Wash well in running tap water to blue.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei and other structures  –  blue to black

Notes

  • This is a modification of Heidenhain’s iron hematoxylin, and differs solely in the concentration of the reagents.
  • The stock solutions are stable for some time.
  • The hematoxylin solution needs to be ripened.
  • The degree of differentiation will determine which tissue components are prominent. This method can demonstrate many structures, including chromosomes, nuclear components, mitochondria and muscle striations
  • The solutions may be reused, with the exception of the solution A used to differentiate, which should be fresh each time.
  • Counterstaining is not recommended.
  • This method is usually recommended for monochrome photography.

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:
    Heidenhain, M., (1892).
    Festschrift Herrn A. von Kolloker zur Feier seines fünfzigjährigen medicinischen
    Doktorjubiläums, p.118. Wilhelm Engellmans, Leipzig, Germany
    and:
    Heidenhain, M.,, (1919).
    Annual report of the Cancer Research Fund, v.16. p.77. London, England.
March 30, 2023
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Masson’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Masson's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematein20 gDye
Potassium alum60 gMordant
Distilled water1 LSolvent
Glacial acetic acid20 mLAcidifier

Compounding Procedure

  1. Dissolve the Alum in boiling water.
  2. Add the dye.
  3. Filter when cool.
  4. Add acetic acid.
  5. The solution may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • This solution uses hematein instead of hematoxylin
  • The high concentration of hematein indicates this is most likely a strong, regressive solution.
  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
March 30, 2023
Love0