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Mordanted Hematoxylin

Cole’s Alum Hematoxylin Variants

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

Cole's Alum Hematoxylin Variants

8
steps
7
materials
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References to Cole’s hematoxylin that do not specify which of the two formulae is meant, usually refer to the 1943 formula.

Materials

MaterialVariantFunction
19031943
Hematoxylin6 g1.5 gDye
Ammonium alum6 g100 gMordant
Distilled water320 mL950 mLSolvent
100% ethanol320 mL50 mLSolvent
Glycerol290 mL–Stabiliser
Iodine–0.5 gOxidant
Glacial acetic acid75 mLSee noteAcidifier

Compounding Procedures

1903

  1. Dissolve the alum in water.
  2. Dissolve the hematoxylin in ethanol.
  3. Combine, then add the other ingredients and mix well.
  4. The solution must ripen before use.

1943

  1. Dissolve the hematoxylin in 250 mL water with heat.
  2. Dissolve the alum in 700 mL water.
  3. Dissolve the iodine in the ethanol.
  4. Combine the dye and iodine solutions.
  5. Add the alum solution.
  6. Bring to a boil.
  7. Cool and filter.
  8. 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

  • The original 1943 formula contains no added acid. Used progressively, and applied for 5-10 minutes, it gives results similar to those obtained with a differentiated regressive formula.
  • The addition of 20 mL glacial acetic acid to the 1943 formula increases nuclear selectivity and extends the working life of the solution. This is often used for progressive nuclear counterstaining and in the celestine blue-hemalum sequence.
  • The staining time for the 1903 formula 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. Culling, C.F.A., Allison, R.T. and Barr, W.T.
    Cellular Pathology Technique, Ed.4.
    Butterworth, London, UK.
  2. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  3. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  4. 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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Böhmer’s Alum Hematoxylin

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

Böhmer's Alum Hematoxylin

15
steps
4
materials
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Böhmer’s formula is the original alum hematoxylin solution for nuclear staining. It is included for educational and historical reasons as the solution has little use in modern histotechnology.

Materials

Solution A

MaterialVariantFunction
Var 1Var 2
Hematoxylin3.5 g8 gDye
100% ethanol100 mL100 mLSolvent

Solution B

MaterialVariantFunction
Var 1Var 2
Ammonium alum0.3 g0.3 gMordant
Distilled water100 mL100 mLSolvent

Compounding procedure

Var 1 is taken from the Microtomist’s Formulary and Guide, and Var 2 from the Microtomist’s Vade-Mecum. The difference in concentration of the hematoxylin may be due to converting an alcoholic logwood extract to grams of dye. In any case, the way it is used makes the differences irrelevant.

Originally, solution A would have been made by soaking logwood chips in ethanol until a suitable concentration of dye was obtained. The solution would then have been allowed to ripen for a long time until it was distinctly deep brown, and filtered before it was used. In a modern variation, simply dissolve the dye in ethanol and leave to ripen, or add a small amount of sodium iodate.

The original called for a few drops of solution A to be added to a small quantity of solution B in a watch glass until the depth of color was judged to be correct. For today’s use, perhaps 5 mL solution A added to 45 mL solution B, more or less, would be satisfactory.

Protocol

Standard Method

  1. Place a small amount of staining solution into a watch glass.
  2. Place frozen sections into the staining solution for an appropriate time.
  3. Transfer sections through at least two changes of clean water.
  4. Mount onto slides.
  5. Dehydrate in ethanol, clear in xylene and mount with a resinous medium

Alternative Method

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  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 appropriate time should be determined by trial, but 10-20 minutes should suffice. The time will depend on the amount of solution A added to solution B. Smaller amounts are likely to take longer to stain with a paler final coloration.
  • 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.
  2. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
March 30, 2023
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Bennett’s Alum Hematoxylin

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

Bennett's Alum Hematoxylin

6
steps
6
materials
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Materials

MaterialAmountFunction
Hematoxylin1 gDye
Potassium alum90 gMordant
Distilled water1 LSolvent
Sodium iodate0.2 gOxidant
Chloral hydrate50 gStabiliser
Citric acid1 gAcidifier

Compounding procedure

  1. Heat the water. Then add in order:
    1. Hematoxylin
    2. Sodium iodate
    3. Alum
    4. Citric acid
    5. Chloral hydrate.

    Note: Dissolve each ingredient before adding the next.

  2. Cool to room temperature and filter. It may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5-20 minutes.
  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

  • Putt gives no information regarding staining time and characteristics, but the similarity of the formula to Mayer’s (Langeron’s) hemalum suggests it is progressive and selectively nuclear. A staining time of 10-20 minutes should be adequate.
  • 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. F. A. Putt
    Manual of Histopathological Staining Methods
    John Wiley & Sons, New York, NY., USA
March 30, 2023
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Bencosme’s Alum Hematein

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

Bencosme's Alum Hematein

8
steps
8
materials
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Materials

Original

MaterialAmountFunction
Hematein2 gDye
Potassium alum133 gMordant
Distilled water920 mLSolvent
Glacial acetic acid20 mLAcidifier

Variant

MaterialAmountFunction
Hematein2.5 gDye
Potassium alum120 gMordant
Distilled water1 LSolvent
Glacial acetic acid10 mLAcidifier

Compounding procedure

  1. Add the alum to the water in a 2 L flask and bring to a boil for 1 minute.
  2. Add the hematein and mix by swirling.
  3. Place an inverted funnel over the flask and simmer for 10 minutes, shaking frequently.
  4. Cool to room temperature and add the acetic acid.
  5. Filter before use and twice weekly.
  6. It may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 2 minutes.
  3. Rinse with water.
  4. Differentiate with 0.5% hydrochloric acid in 70% ethanol for one second.
  5. Rinse well with water and blue.
  6. Rinse well with water.
  7. Counterstain with HPS.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  shades of red and yellow.

Notes

  • Note that this solution uses hematein rather than hematoxylin. Hematoxylin could be used, but would require 0.4 grams sodium iodate to be added along with the dye for complete oxidation in the original solution, although 0.3 grams might be more appropriate to extend the life. Comparable amounts would be 0.5 g and 0.4 g, respectively, for the variant formula.
  • When freshly made this solution stains in 2 minutes. Staining time slowly increases to 10 minutes as the solution ages. Life is about a month.
  • 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. Medical Laboratory Technology, 2nd ed. (1969)
    Lynch M.J., Raphael S.S., Mellor L.D., Spare P.D. and Inwood M.J.H.,
    W. B. Saunders Co., Toronto, On., Canada
March 30, 2023
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Bensley’s Copper Hematoxylin

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

Bensley's Copper Hematoxylin

10
steps
8
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin,1 gDye
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Cupric acetate,to saturationMordant
Distilled water100 mLSolvent

Solution C

MaterialAmountFunction
Potassium chromate,5 g–
Distilled water100 mLSolvent

Solution D

MaterialAmountFunction
Weigert’s borax-ferricyanide20 mLDifferentiator
Distilled water80 mLDiluent

Compounding procedure

Make each solution separately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 5 minutes.
  3. Rinse with water.
  4. Dip into solution B.
  5. Rinse with water.
  6. Place into solution C for 2 minutes.
  7. Return to solution A for 1 minute.
  8. Place in solution D until differentiated.
  9. Rinse well with water.
  10. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei and other structures  –  blue-black

Notes

  • After step 7, the sections should be intense black. Repeat steps 5-7 if necessary.
  • Bensley recommended prestaining with Mayers mucicarmine.
  • Best results are obtained with freshly made solutions

Safety Note

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

References

  1. Bensley R. R. and Bensley, S. H., (1938)
    Handbook of Histological and Cytological Technique.
    U. Chicago Press, Chicago, USA
March 30, 2023
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Bosma’s Alum Hematoxylin

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

Bosma's Alum Hematoxylin

8
steps
6
materials
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Materials

MaterialAmountFunction
Hematoxylin, 10% alc.25 mLDye
Ammonium alum25 gMordant
Tap water865 mLSolvent
Diethylene glycol100 mLStabiliser
Sodium iodate0.2 gOxidant
Glacial acetic acid10 mLAcidifier

Compounding procedure

  1. Place 815 mL hot tap water in a large flask.
  2. Add the ammonium alum, mix well to dissolve and cool to room temperature.
  3. Add the alcoholic hematoxylin solution (ripened).
  4. Add the sodium iodate dissolved in 50 mL cold tap water and mix well.
  5. Add the diethylene glycol and mix well.
  6. Add the acetic acid and mix well. The pH should be 3.1 to 3.3 and 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

  • 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. Bosma, Rob
    A useful hematoxylin without toxic chemicals.
    Histologic, V 18, Nº 1, January 1988
March 30, 2023
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Bullard’s Alum Hematoxylin

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

Bullard's Alum Hematoxylin

8
steps
10
materials
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Materials

MaterialAmountFunction
Hematoxylin8 gDye
Ammonium alum20 gMordant
Distilled water250 mLSolvent
50% ethanol144 mLSolvent
Glacial acetic acid16 mLAcidifier
Mercuric oxide, red8 gOxidant
95% ethanol275 mLSolvent
Glycerol330 mLStabiliser
Glacial acetic acid18 mLAcidifier
Ammonium alum40 gMordant

Compounding procedure

  1. Combine the first five ingredients and bring to a boil.
  2. Add the mercuric oxide with caution.
  3. Cool and filter.
  4. Add the last four ingredients.
  5. The solution may be used immediately, and is stable for more than a year.

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 solution is likely regressive
  • The appropriate 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. F. A. Putt
    Manual of Histopathological Staining Methods
    John Wiley & Sons, New York, NY., USA
March 30, 2023
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Carazzi’s Alum Hematoxylin

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

Carazzi's Alum Hematoxylin

6
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Potassium alum50 gMordant
Distilled water800 mLSolvent
Glycerol200 mLStabiliser
Sodium iodate0.2 gOxidant

Compounding procedure

  1. Dissolve the hematoxylin in the glycerol.
  2. Dissolve the alum in 750 mL of the water.
  3. Dissolve the sodium iodate in the remaining 50 mL water.
  4. Add the alum solution to the hematoxylin solution slowly, while mixing well.
  5. Add the sodium iodate solution. Mix well.
  6. Filter.
  7. The solution may be used immediately, and is stable for about six months.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5 minutes.
  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

  • This is a progressive solution giving little background staining.
  • Doubling the hematoxylin to 2 g intensifies nuclear staining.
  • The double strength solution is recommended for frozen sections with about 1 minute staining time.
  • 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.
  • In the original paper, 0.02 grams sodium iodate was specified. This would oxidize only a small part of the hematoxylin. The amount specified above would permit full oxidation.

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. (1982)
    Theory and practice of histological techniques, Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  2. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide. p.187.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Smith’s Vanadium Hematoxylin

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

Smith's Vanadium Hematoxylin

7
steps
5
materials
print

When differentiated with dilute lithium carbonate, this solution demonstrates basic proteins, including nuclear histones.

Materials

MaterialAmountFunction
Hematoxylin50 mgDye
Ethanol, 100%5 mLSolvent
Glycerol10 mLSolvent
Distilled water35 mLSolvent
Ammonium metavanadate200 mgSolvent

Compounding procedure

  1. Dissolve the hematoxylin in the ethanol, then add the glycerol and water in sequence.
  2. Add the ammonium vanadate and stir for 30 minutes.
  3. The ammonium vanadate may not completely dissolve. The pH is 6.2.

Tissue Sample

Paraffin sections of Schaudinn, Bouin or formalin fixed tissues are suitable. Sections of Zenker or Helly fixed tissues must be soaked in saturated aqueous lithium carbonate for 4 hours to stain satisfactorily.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the vanadate hematoxylin for 30 minutes.
  3. Place directly into 0.08% aqueous lithium carbonate for the appropriate time.
    Differentiation times in lithium carbonate

    FixativeTime
    Bouin2 minutes
    Formalin, 10%2 minutes
    Helly10 minutes
    Schaudinn5 minutes
    Zenker10 minutes
  4. Briefly rinse with distilled water for 2 seconds.
  5. Optionally, counterstain if wished.
  6. Dehydrate with 70%, 95% and absolute ethanols.
  7. Clear with xylene and mount with a resinous medium (Permount specified).

Expected Results

  • Nuclear histone  –  blue
  • Ribosomal protein  –  paler blue
  • Other protein  –  as counterstain or unstained

Notes

  • Counterstaining can be accomplished at step 5 with 1% aqueous eosin Y, phloxine B or erythrosin B.
  • Alternatively, metanil yellow at 0.1% in 95% ethanol containing 0.1% acetic acid may be used between the 70% and 95% ethanols in step 6.
  • Counterstaining with 0.01% aqueous safranin O stained RNA intensely, giving good contrast between blue nuclear histone and endoplasmic reticulum.

Safety Note

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

References

  1. Smith, A. A., (1995)
    A vanadate hematoxylin stain for basic proteins.
    Biotechnic and Histochemistry, v. 70, Nº 5, p. 5.
March 30, 2023
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Baker’s Hematal Variants

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

Baker's Hematal Variants

15
steps
11
materials
print

Materials

MaterialSolutionFunction
Stock AStock B
Aluminum sulphate15.76 g–Mordant
Distilled water1 L–Solvent
Haematein–1.876 gDye
Ethylene glycol, 50%, aqu.–1 LSolvent
MaterialHematal-8Hematal-16
Stock A1 vol2 vol
Stock B1 vol1 vol
Ethylene glycol, 50%, aqu.–1 vol
MaterialSolutionFunction
Standard AStandard B
Aluminum sulphate3.94 g–Mordant
Distilled water1 L–Solvent
Haematoxylin–5 gDye
Sodium iodate–0.5 gDye
Distilled water–1 LSolvent
Sulphuric acid, 0.5% aqueousas needed
Ammonia, 0.5% aqueousas needed

Compounding Procedures

Hematal-8 and Hematal-16 Solutions

  1. Prepare each stock solution separately.
  2. Combine as specified.
  3. The solution may be used immediately.

Standard Solution

  1. Prepare each solution separately.
  2. Standard solution B should be brought to a boil, then immediately cooled.

Protocol

Hematal-8 & Hematal-16 Solutions

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for the specified time.
    • Hematal-8 for 2-5 minutes.
    • Hematal-16 for 10 minutes.
  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.

Standard Solutions

  1. Bring sections to water with xylene and ethanol.
  2. Place into standard solution A for 1 hour.
  3. Rinse with water.
  4. Place into standard solution B for 20 minutes.
  5. Rinse with water.
  6. Place into 0.5% sulphuric acid for 40 seconds.
  7. Rinse with water.
  8. Place into 0.5% ammonia water for 5 seconds.
  9. Wash well with water.
  10. Optionally, counterstain with eosin.
  11. 70% ethanol for 30 seconds.
  12. 90% ethanol for 30 seconds.
  13. Absolute ethanol, 2 changes, 1 minute each.
  14. Xylene, 2 changes, 1 minute each.
  15. Mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • Hematal-8 and Hematal-16 may be used for routine staining. The solutions are quickly and conveniently prepared.
  • Hematal-8 may be used regressively by applying for 30 minutes, then lightly differentiating in acid. Dilute sulphuric acid was recommended (0.5% aqu.), but hydrochloric acid was usable.
  • Hematal-8 has 8 atoms of aluminum for each molecule of hematein, Hematal-16 has 16, hence the names.
  • 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.
  • The standard method was used to give consistent staining with the usually expected appearance, but with the ability to vary each step for experimental reasons. It was not intended for routine use.

Safety Note

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

References

  1. Baker, J. R., (1962),
    Experiments on the action of mordants: 2. Aluminium-haematein.
    Quarterly Journal of Microscopical Science, v. 103, pt. 4, pp. 493-517.
March 30, 2023
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