Heidenhain’s iron hematoxylin technique uses two separate solutions for staining. First, an iron mordant is applied, usually ferric ammonium sulfate or ferric chloride. This is followed by a hematoxylin solution. The overstained preparations are then differentiated in the iron mordant solution, often diluted, or in some similar solution. Numerous variations on this theme have been published, most of them staining very similarly, as they differ largely in the concentration of the reagents and the times for which they are applied.
In the chart below it is to be understood that all solids are in grams and all fluids in milliliters. The amounts given are to be dissolved in 100 milliliters of the solvent for each solution. The solvent is distilled water unless otherwise stated.
The term visual in the Differentiate column means to control differentiation microscopically.
| Variant | Solution (in distilled water unless otherwise stated) | Time | Differentiate with solution | Comment | |||
|---|---|---|---|---|---|---|---|
| A | B | C | A | B | |||
| Heidenhain | Iron alum, 2.5% | Hematoxylin, 0.5% | – | ½ – 24 hrs. | ½ – 24 hrs. | A, visual | – |
| Bütschli | Ferric acetate, 2% | Hematoxylin, 0.5% | – | 24 hrs | 3 hrs | – | For protozoa |
| Diamond | Iron alum, 4% | Hematoxylin, 0.5% in distilled water with 0.1% tergitol | Picric acid, saturated in absolute ethanol | 5 min. | 5 min. | C, 3-5 min. | – |
| Dobell | Iron alum, 1% in 70% ethanol | Hematein, 1% in 70% ethanol | Hydrochloric acid, 0.1% in 70% ethanol | 10 min | 10 min | C, visual | For protozoa |
| Freitas | Iron alum, 0.5% in 70% ethanol | Hematein, 1% in 60 mL 70% ethanol and 30 mL phosphate buffer pH 7.6 | Picric acid, 0.3% in 70% ethanol | 1 hr | 1 hr | C, visual | – |
| French | Iron alum, 3.5% in 70% ethanol | Hematoxylin, 1% in 98 mL 95% ethanol and 2 mL saturated aqueous lithium carbonate | Iron alum, 1% | Overnight | Overnight | C, visual | – |
| Galiano | Iron alum, 3% | Hematoxylin, 0.2% in acetic acid, 20% aqueous | Eosin, 1.5% in acetic acid, 25% aqueous | 15 mins | until dark | C, visual | Rinse with 0.1% ammonia in 95% ethanol after solution C |
| Haggquist | Iron alum, 5% | Hematoxylin, 1% | Ferric chloride, 1% | 1 hr | 1 hr | C, visual | – |
| Hance | Iron alum, 2.5% | 10% ethanolic hematoxylin, 10 mL in 100 mL distilled water with sodium bicarbonate | – | ½ – 2 hrs | 30 min | C, visual | Add SB until sol B changes from yellow to a plum color |
