Kleinenberg's Alum Hematoxylin
There are two alternate formulae for Kleinenberg’s Alum hematoxylin.
Materials
Formula I
Hematoxylin
Material | Amount | Function |
---|---|---|
Hematoxylin | saturated | Dye |
100% ethanol | 100 mL | Solvent |
Alum
Material | Amount | Function |
---|---|---|
Ammonium alum | saturated | Mordant |
70% ethanol | 100 mL | Solvent |
Calcium
Material | Amount | Function |
---|---|---|
Calcium chloride | saturated | Mordant |
70% ethanol | 100 mL | Solvent |
Compounding Procedure
- Prepare each of the solutions.
- For use, combine:
- Alum solution – 85 mL
- Calcium solution – 15 mL
- Hematoxylin solution – 1 mL
Formula II
Hematoxylin
Material | Amount | Function |
---|---|---|
Hematoxylin | saturated | Dye |
100% ethanol | 100 mL | Solvent |
Alum A
Material | Amount | Function |
---|---|---|
Ammonium alum | saturated | Mordant |
Calcium chloride | saturated | Mordant |
70% ethanol | 100 mL | Solvent |
Alum B
Material | Amount | Function |
---|---|---|
Potassium alum | saturated | Mordant |
70% ethanol | 88 mL | Solvent |
Alum A solution | 12 mL | – |
Compounding Procedure
- Prepare each of the solutions.
- For use, combine:
- Alum B solution – 100 mL
- Hematoxylin solution – 3 mL
Protocol
- Bring sections to water with xylene and ethanol.
- Place into the staining solution for an appropriate time.
- Rinse well with water.
- Differentiate with acid ethanol if necessary.
- Rinse with water and blue.
- Rinse well with water.
- Counterstain if desired.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – blue
- Background – as counterstain or unstained
Notes
- Although not stated, the saturated hematoxylin solution should presumably be allowed to ripen.
- Formula I uses 1 mL of the saturated hematoxylin solution, whereas formula II uses 3 mL, i.e. it is considerably stronger. This may affect the staining times.
- Alum and calcium chloride combined give aluminum chloride 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
- Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Cole, A. C., (1884).
The methods of microscopical research. (formula I), and
Böhm, A. & Opel, A., (1907).
Manuel de technique microscopique, Ed. 4, (formula II).