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Kleinenberg’s Alum Hematoxylin

Kleinenberg's Alum Hematoxylin

8
steps
5
materials

There are two alternate formulae for Kleinenberg’s Alum hematoxylin.

Materials

Formula I

Hematoxylin

MaterialAmountFunction
HematoxylinsaturatedDye
100% ethanol100 mLSolvent

Alum

MaterialAmountFunction
Ammonium alumsaturatedMordant
70% ethanol100 mLSolvent

Calcium

MaterialAmountFunction
Calcium chloridesaturatedMordant
70% ethanol100 mLSolvent

Compounding Procedure

  1. Prepare each of the solutions.
  2. For use, combine:
    1. Alum solution – 85 mL
    2. Calcium solution – 15 mL
    3. Hematoxylin solution – 1 mL

Formula II

Hematoxylin

MaterialAmountFunction
HematoxylinsaturatedDye
100% ethanol100 mLSolvent

Alum A

MaterialAmountFunction
Ammonium alumsaturatedMordant
Calcium chloridesaturatedMordant
70% ethanol100 mLSolvent

Alum B

MaterialAmountFunction
Potassium alumsaturatedMordant
70% ethanol88 mLSolvent
Alum A solution12 mL

Compounding Procedure

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

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

  • 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

  1. 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).