Baker's Hematal Variants
Materials
| Material | Solution | Function | |
|---|---|---|---|
| Stock A | Stock B | ||
| Aluminum sulphate | 15.76 g | – | Mordant |
| Distilled water | 1 L | – | Solvent |
| Haematein | – | 1.876 g | Dye |
| Ethylene glycol, 50%, aqu. | – | 1 L | Solvent |
| Material | Hematal-8 | Hematal-16 |
|---|---|---|
| Stock A | 1 vol | 2 vol |
| Stock B | 1 vol | 1 vol |
| Ethylene glycol, 50%, aqu. | – | 1 vol |
| Material | Solution | Function | |
|---|---|---|---|
| Standard A | Standard B | ||
| Aluminum sulphate | 3.94 g | – | Mordant |
| Distilled water | 1 L | – | Solvent |
| Haematoxylin | – | 5 g | Dye |
| Sodium iodate | – | 0.5 g | Dye |
| Distilled water | – | 1 L | Solvent |
| Sulphuric acid, 0.5% aqueous | as needed | ||
| Ammonia, 0.5% aqueous | as needed | ||
Compounding Procedures
Hematal-8 and Hematal-16 Solutions
- Prepare each stock solution separately.
- Combine as specified.
- The solution may be used immediately.
Standard Solution
- Prepare each solution separately.
- Standard solution B should be brought to a boil, then immediately cooled.
Protocol
Hematal-8 & Hematal-16 Solutions
- Bring sections to water with xylene and ethanol.
- Place into the staining solution for the specified time.
- Hematal-8 for 2-5 minutes.
- Hematal-16 for 10 minutes.
- Rinse with water and blue.
- Rinse well with water.
- Counterstain if desired.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Standard Solutions
- Bring sections to water with xylene and ethanol.
- Place into standard solution A for 1 hour.
- Rinse with water.
- Place into standard solution B for 20 minutes.
- Rinse with water.
- Place into 0.5% sulphuric acid for 40 seconds.
- Rinse with water.
- Place into 0.5% ammonia water for 5 seconds.
- Wash well with water.
- Optionally, counterstain with eosin.
- 70% ethanol for 30 seconds.
- 90% ethanol for 30 seconds.
- Absolute ethanol, 2 changes, 1 minute each.
- Xylene, 2 changes, 1 minute each.
- Mount with a resinous medium.
Expected Results
- Nuclei – blue
- Background – as counterstain or unstained
Notes
- Hematal-8 and Hematal-16 may be used for routine staining. The solutions are quickly and conveniently prepared.
- Hematal-8 may be used regressively by applying for 30 minutes, then lightly differentiating in acid. Dilute sulphuric acid was recommended (0.5% aqu.), but hydrochloric acid was usable.
- Hematal-8 has 8 atoms of aluminum for each molecule of hematein, Hematal-16 has 16, hence the names.
- 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.
- The standard method was used to give consistent staining with the usually expected appearance, but with the ability to vary each step for experimental reasons. It was not intended for routine use.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Baker, J. R., (1962),
Experiments on the action of mordants: 2. Aluminium-haematein.
Quarterly Journal of Microscopical Science, v. 103, pt. 4, pp. 493-517.
