Slidders' Alum Hematoxylin
This formulation differs from most others in that it is made from three stock solutions. It contains no added acid, relying on the high alum:dye ratio for it’s progressive staining. Consequently, the carry over of tap water fairly rapidly causes both dilution and raises the pH. Due to these, it has a shorter working life than many other formulations. It was designed to be quickly prepared in laboratories that stain large numbers of slides and prefer to use fresh solution daily, but which would like to avoid the poor background staining obtained with formulations that incorporate an acid to accentuate nuclei.
Materials
Stock mordant
| Material | Amount | Function |
|---|---|---|
| Potassium alum | 2 kg | Mordant |
| Distilled water | 19.8 L | Solvent |
| Strong formalin | 200 mL | Preservative |
Stock hematoxylin
| Material | Amount | Function |
|---|---|---|
| Hematoxylin | 20 g | Dye |
| Strong formalin | 200 mL | Solvent |
Stock oxidizer
| Material | Amount | Function |
|---|---|---|
| Sodium iodate | 4 g | Oxidant |
| Distilled water | 200 mL | Solvent |
Compounding Procedure
Stock mordant
- Dissolve the Alum into 4 litres of distilled water, using heat.
- Add the balance of the water and the formalin.
Stock hematoxylin and stock oxidizer
- Dissolve each constituent into the appropriate solvent.
Working solution
- Place 400 mL of the stock mordant into a 2 litre flask.
- Add 20 mL stock hematoxylin and 20 mL stock oxidizer.
- Under a fume hood, bring to the boil, then remove immediately.
- Add 1600 mL stock mordant.
- The solution may be used immediately.
The formulated solution is stable for about 2 months at room temperature. The stock solutions are stable at least 6 months at room temperature.
The solution may also be made in smaller volumes according to a more traditional regimen:
| Material | Amount | Function |
|---|---|---|
| Hematoxylin | 1 g | Dye |
| Potassium alum | 100 g | Mordant |
| Distilled water | 1 L | Solvent |
| Sodium iodate | 0.2 g | Oxidant |
Compounding Procedure
- Combine the ingredients.
- Bring to the boil and immediately remove from heat.
- Cool to room temperature.
- The solution may be used immediately after it has cooled.
Protocol
- Bring sections to water with xylene and ethanol.
- Place into the staining solution for about 5 minutes.
- 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
- The formalin used as the solvent in the stock Alum and hematoxylin solutions should be concentrated, i.e. 37-40% aqueous formaldehyde. It acts as both a preservative and an anti-oxidant.
- 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
- Slidders, W. (1988)
Preparation of Alum hematoxylin.
Medical Laboratory Sciences, v 45, pp. 405.
