Debiden's
Alum Hematoxylin Variants

Ingredient	1980	1991	Function
Hematoxylin	5 g	5 g	Dye
Potassium alum	100 g	100 g	Mordant
Distilled water	1 L	1 L	Solvent
Glacial acetic acid	20 mL	20 mL	Acidifier
Mercuric oxide, red	2.5 g	–	Oxidant
Javex (5.25% sodium hypochlorite)	–	2 mL	Oxidant

 

Compounding procedure (Both)
Dissolve the alum in the water in a large flask.
Add the hematoxylin. Mix well.
Place in a 60-65°C oven overnight.
Remove from the oven, and add the appropriate oxidising agent.
Stir until cold. Filter.

1980
Acetic acid may be added as soon as it is cooled following preparation.
It is usable immediately.
It is preferable to prepare small volumes monthly.

1991
This improves in staining ability if allowed to ripen for a week or two.
If acetic acid is used it should be added just before use.

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

• Nuclei  –  blue
• Background  –  as counterstain or unstained

Notes

1. These two solutions are modifications of Harris' hematoxylin and are regressive.
2. Paraffin sections should be stained for 5 minutes, and cytology smears for 45 seconds.
3. As with many strong regressive formulae, mucin may be stained blue.
4. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
5. 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

Debiden, D., (1987)
Improved preparation of Harris' hematoxylin
Histologic, v.18, No.8

Debiden, D., (1991)
A new oxidant for Harris' hematoxylin
Histologic, v.21, No.2