De Groot's Alum Hematoxylin
Materials
| Material | Amount | Function |
|---|---|---|
| Hematoxylin | 2 g | Dye |
| Ammonium alum | 22 g | Mordant |
| Distilled water | 270 mL | Solvent |
| 95% ethanol | 650 mL | Solvent |
| Glycerol | 80 mL | Stabiliser |
| Hydrogen peroxide | 7.5 mL | Oxidant |
| Potassium ferricyanide | 0.8 g | – |
| Calcium chloride | 15 g | – |
| Sodium bromide | 7.5 g | – |
Compounding procedures
- Mix the ethanol, water and glycerol to make the solvent.
- Add the peroxide to 15 mL of the solvent.
- Add the hematoxylin, and dissolve.
- Dissolve the calcium chloride and sodium bromide in 250 mL of the solvent.
- Mix with the hematoxylin solution.
- Add half the alum, and dissolve.
- Dissolve the potassium ferricyanide in 400 mL of the solvent.
- Add to the hematoxylin solution.
- Dissolve the remaining alum in the remaining solvent.
- Add to the hematoxylin solution.
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
- Both hydrogen peroxide and potassium ferricyanide are oxidizing agents. However, it is not clear if potassium ferricyanide is present for this reason.
- The strength of hydrogen peroxide is not specified, but the commonest laboratory strength is 30 vols.
- Although calcium can mordant hematoxylin, it is not clear if it is present for that reason.
- The purpose of the sodium bromide is not clear. It may be present as a preservative, similar to chloral hydrate in some other formulas.
- 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.
