Molnar's Alum Hematoxylin Variants
Materials
| Material | Variation I | Variation I | Function |
|---|---|---|---|
| Hematoxylin | 10 g | 4 g | Dye |
| Ammonium or potassium alum | 50 g | 50 g | Mordant |
| Distilled water | 1 L | 1 L | Solvent |
| 95% ethanol | 50 mL | Solvent | |
| Mercuric oxide | 5 g | Oxidant | |
| Sodium iodate | 0.3 g | Oxidant | |
| Glacial acetic acid | 20 mL | Acidifier | |
| Citric acid | 1.5 g | Acidifier | |
| Chloral hydrate | 75 g | Antioxidant |
Compounding Procedures
Variation I
- Dissolve the dye into the ethanol, and the Alum into the water.
- Combine the solutions in an Erlenmeyer flask and slowly add the mercuric oxide
- Reheat until the solution changes colour to a dark purple.
- Remove from heat and cool rapidly by swirling the flask in cold water.
- When cool, add the acetic acid.
Variation II
- Dissolve the Alum into the water.
- Add the hematoxylin, sodium iodate, citric acid and chloral hydrate in that order.
- Filter before use.
Protocol
- Bring sections to water with xylene and ethanol.
- Place into the staining solution for the specified time.
- Var I for 10 minutes.
- Var II for 8-10 seconds.
- Rinse well with water.
- Differentiate Var I with acid ethanol for a few seconds.
- 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
- Var I was recommended for double embedded tissues.
- The use of mercuric oxide as an oxidant is now deprecated due to its toxicity. Var I could be modified by using sodium iodate at about 0.3 grams instead.
- Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
- 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
- Molnar, L. N.,
Modification of Harris hematoxylin for sections from tissue double embedded with nitrocellulose and paraffin.
Histologic, v 5, Nº 1, January, 1975 - Molnar, L. N.,
Modification of Mayer’s hematoxylin-eosin method.
Histologic, v 6, Nº 4, October, 1976
