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Heidenhain’s Iron Hematoxylin Variants

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.

VariantSolution (in distilled water unless otherwise stated)TimeDifferentiate with solutionComment
HeidenhainIron alum, 2.5%Hematoxylin, 0.5%½ – 24 hrs.½ – 24 hrs.A, visual
BütschliFerric acetate, 2%Hematoxylin, 0.5%24 hrs3 hrsFor protozoa
DiamondIron alum, 4%Hematoxylin, 0.5% in distilled water with 0.1% tergitolPicric acid, saturated in absolute ethanol5 min.5 min.C, 3-5 min.
DobellIron alum, 1% in 70% ethanolHematein, 1% in 70% ethanolHydrochloric acid, 0.1% in 70% ethanol10 min10 minC, visualFor protozoa
FreitasIron alum, 0.5% in 70% ethanolHematein, 1% in 60 mL 70% ethanol and 30 mL phosphate buffer pH 7.6Picric acid, 0.3% in 70% ethanol1 hr1 hrC, visual
FrenchIron alum, 3.5% in 70% ethanolHematoxylin, 1% in 98 mL 95% ethanol and 2 mL saturated aqueous lithium carbonateIron alum, 1%OvernightOvernightC, visual
GalianoIron alum, 3%Hematoxylin, 0.2% in acetic acid, 20% aqueousEosin, 1.5% in acetic acid, 25% aqueous15 minsuntil darkC, visualRinse with 0.1% ammonia in 95% ethanol after solution C
HaggquistIron alum, 5%Hematoxylin, 1%Ferric chloride, 1%1 hr1 hrC, visual
HanceIron alum, 2.5%10% ethanolic hematoxylin, 10 mL in 100 mL distilled water with sodium bicarbonate½ – 2 hrs30 minC, visualAdd SB until sol B changes from yellow to a plum color