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

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).

 


 

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