Böhmer's Alum Hematoxylin
Böhmer’s formula is the original alum hematoxylin solution for nuclear staining. It is included for educational and historical reasons as the solution has little use in modern histotechnology.
Materials
Solution A
| Material | Variant | Function | |
|---|---|---|---|
| Var 1 | Var 2 | ||
| Hematoxylin | 3.5 g | 8 g | Dye |
| 100% ethanol | 100 mL | 100 mL | Solvent |
Solution B
| Material | Variant | Function | |
|---|---|---|---|
| Var 1 | Var 2 | ||
| Ammonium alum | 0.3 g | 0.3 g | Mordant |
| Distilled water | 100 mL | 100 mL | Solvent |
Compounding procedure
Var 1 is taken from the Microtomist’s Formulary and Guide, and Var 2 from the Microtomist’s Vade-Mecum. The difference in concentration of the hematoxylin may be due to converting an alcoholic logwood extract to grams of dye. In any case, the way it is used makes the differences irrelevant.
Originally, solution A would have been made by soaking logwood chips in ethanol until a suitable concentration of dye was obtained. The solution would then have been allowed to ripen for a long time until it was distinctly deep brown, and filtered before it was used. In a modern variation, simply dissolve the dye in ethanol and leave to ripen, or add a small amount of sodium iodate.
The original called for a few drops of solution A to be added to a small quantity of solution B in a watch glass until the depth of color was judged to be correct. For today’s use, perhaps 5 mL solution A added to 45 mL solution B, more or less, would be satisfactory.
Protocol
Standard Method
- Place a small amount of staining solution into a watch glass.
- Place frozen sections into the staining solution for an appropriate time.
- Transfer sections through at least two changes of clean water.
- Mount onto slides.
- Dehydrate in ethanol, clear in xylene and mount with a resinous medium
Alternative Method
- Bring sections to water via xylene and ethanol.
- Place into the staining solution for an appropriate time.
- 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 appropriate time should be determined by trial, but 10-20 minutes should suffice. The time will depend on the amount of solution A added to solution B. Smaller amounts are likely to take longer to stain with a paler final coloration.
- 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
- 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.
