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.

Stock mordant	Amount	Function
Potassium alum	2 kg	Mordant
Distilled water	19.8 L	Solvent
Strong formalin	200 mL	Preservative
Stock hematoxylin	Amount	Function
Hematoxylin	20 g	Dye
Strong formalin	200 mL	Solvent
Stock oxidiser	Amount	Function
Sodium iodate	4 g	Oxidant
Distilled water	200 mL	Solvent

 

Compounding procedure
Stock mordant
Dissolve the alum into 4 litres distilled water, using heat.
Add the balance of the water and the formalin.
 
Stock hematoxylin and stock oxidiser
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 oxidiser.
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:–

Ingredient	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 it has cooled.

Method

1. Bring sections to water with xylene and ethanol.
2. Place into the staining solution for about 5 minutes.
3. Rinse with water and blue.
4. Rinse well with water.
5. Counterstain if desired.
6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  blue
• Background  –  as counterstain or unstained

Notes

1. 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.
2. 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
Slidders, W. (1988)
Preparation of alum hematoxylin.
Medical Laboratory Sciences, v 45, pp. 405.