Kleinenberg's
Alum Hematoxylin

There are two alternate formulae for Kleinenberg's alum hematoxylin.

Formula I
Hematoxylin	Amount	Function
Hematoxylin	saturated	Dye
100% ethanol	100 mL	Solvent
Alum	Amount	Function
Ammonium alum	saturated	Mordant
70% ethanol	100 mL	Solvent
Calcium	Amount	Function
Calcium chloride	saturated	Mordant
70% ethanol	100 mL	Solvent

Formula II
Hematoxylin	Amount	Function
Hematoxylin	saturated	Dye
100% ethanol	100 mL	Solvent
Alum A	Amount	Function
Ammonium alum	saturated	Mordant
Calcium chloride	saturated	Mordant
70% ethanol	100 mL	Solvent
Alum B	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 solution – 85 mL
Calcium solution – 15 mL
Hematoxylin solution – 1 mL

Compounding procedure
Prepare each of the solutions.
For use, combine:–
Alum B solution – 100 mL
Hematoxylin solution – 3 mL

Method

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

1. Although not stated, the saturated hematoxylin solution should presumably be allowed to ripen.
2. 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.
3. Alum and calcium chloride combined give aluminum chloride hematoxylin.
4. The staining time should be determined by trial.
5. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
6. 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.

 

Reference
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).