Anderson's
Alum Hematoxylin

Although there are three variations listed for Anderson's formulas, those marked as 1923a and 1923b are variations of the same solution, the former being that given in the Microtomist's Formulary and Guide and the latter in the Microtomist's Vade Mecum.

Ingredient	1923a	1923b	1929	Function
Hematoxylin	2.5 g	2.5 g	5 g	Dye
Ammonium alum	20 g	Sat.	30 g	Mordant
Distilled water	900 mL	900 mL	700 mL	Solvent
95% ethanol	50 mL	50 mL	50 mL	Solvent
Calcium hypochlorite	4 g	–	40 g	Oxidant
Chloramine T or lime chloride		4 g		Oxidant
Glacial acetic acid	50 mL	50 mL	50 mL	Acidifier

 

Compounding procedure

1923a & 1929
Dissolve the calcium hypochlorite in 200 mL water.
Dissolve the hematoxylin in some of the water.
After 4 hours, combine the solutions.
Dissolve the other ingredients in the rest of the water.
Combine with the hematoxylin-hypochlorite solution.

1923b
Bring 700 mL water to the boil, then saturate it with alum (see notes).
Allow to cool for one day, then filter
Dissolve the chloramine T or lime chloride into 200 mL of water.
Leave four hours. Shake occasionally.
Dissolve the hematoxylin in the ethanol
Add the oxidant solution to the hematoxylin.
Mix for a few seconds. It should be dark brown.
Slowly add to the 700 mL alum solution, while shaking.
Add the acetic acid.
It is ready immediately.

Method

1. Bring sections to water with xylene and ethanol.
2. Place into the staining solution for 2-3 minutes.
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 1923a formula oxidises 2.5 grams hematoxylin with 4 grams calcium hypochlorite.
   This is a ratio of 1.6:1.
2. The 1929 formula oxidises 5 grams hematoxylin with 40 grams calcium hypochlorite.
   This is a ratio of 8:1.
3. The 1923b formula is an alternate formula given by Bolles-Lee. It differs from the 1923a formula by using a saturated alum solution, and chloramine T or "commercial chloride of lime" (a crude preparation of calcium chloride with other substances present) as oxidant.
4. The 1923b formula calls for 700 mL saturated aqueous alum. The instructions specify that the water should be saturated at boiling, then cooled to room temperature. This would require about 350 grams alum (at 500 mg/mL), but at room temperature the solution would contain only about 100 grams (at 150 mg/mL). Dissolving 110 grams alum in 700 mL hot water, cooling and filtering would give the same solution.
5. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
6. 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
Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.

Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist's Vade-Mecum. 11 ed.,
Churchill, London, UK.
  Citing:–
    Anderson, J., (1923)
    Journal of Pathology and Bacteriology.

Susan Budavari, Editor, (1996)
The Merck Index, Ed. 12
Merck & Co., Inc., Whitehouse Station, NJ, USA