Bullard's
Alum Hematoxylin

Ingredient	Amount	Function
Hematoxylin	8 g	Dye
Ammonium alum	20 g	Mordant
Distilled water	250 mL	Solvent
50% ethanol	144 mL	Solvent
Glacial acetic acid	16 mL	Acidifier
Mercuric oxide, red	8 g	Oxidant
95% ethanol	275 mL	Solvent
Glycerol	330 mL	Stabiliser
Glacial acetic acid	18 mL	Acidifier
Ammonium alum	40 g	Mordant

 

Compounding procedure
Combine the first five ingredients and bring to the boil.
Add the mercuric oxide with caution.
Cool and filter.
Add the last four ingredients.
The solution may be used immediately, and is stable for more than a year.

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. The solution is likely regressive
li>The appropriate time should be determined by trial.
2. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
3. 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
F. A. Putt
Manual of Histopathological Staining Methods
John Wiley & Sons, New York, NY., USA