Hematoxylin

HEMATOXYLIN, a coloring substance widely used in histological technique. It is obtained from the extract of the logwood tree (Haematoxylon campechianum, Caesalpinaceae, Mexico, Central America). Composition: C₁₆H₁₄O₆ (Chevreuil, 1810; Erdmann, 1842). It forms colorless or yellowish crystals: with 1 molecule of water, rhombic; with 3 molecules of water, tetragonal. The latter are more commonly found on the market with a molecular weight of 302 + 54 = 356 (actual 355.16). Solubility: weak in cold water—0.7177 g in 100 cubic centimeters at 15°; good in hot water, alcohol, and ether. Hematoxylin melts above 100°. Rotation of the polarization plane to the right. In an ammoniacal solution, hematoxylin is oxidized in the presence of oxygen into hematein according to the formula: C₁₆H₁₄O₆ + O = C₁₆H₁₂O₆ + H₂O. Hematoxylin

Hematein (Erdmann, 1842; see Haematein). Slow oxidation of hematoxylin occurs already upon heating. After cooling, solution 1 is added to solution 2. A dark violet liquid is obtained. Boil for 1 minute, cool rapidly; without filtering, pour to the brim into a bottle with a ground-glass stopper. Filter before staining. Stain from 1 minute to 24 hours. Differentiate in 2–3% H₂SO₄ or 0.5–0.1% CH₃COOH. Very black tones, sharp nuclear staining. Do not fade.—b) Two-moment method—Heidenhain's iron hematoxylin (1894). It is also based on the formation of a lake, but differs in that the preparation is first treated (mordanted) with an iron salt, and then stained with an aqueous solution of hematoxylin. Method of staining: 1. Mordanting of sections in a 2.5% aqueous solution of iron-ammonium alum (crystals are violet, the solution is brownish-yellow) for 3–12 hours. 2. Staining for 12–24 hours in an aqueous solution of hematoxylin (4–6 week-old 0.5% solution of Haematoxyl. puriss. in water; or Haematoxyl. puriss. 1.0, Ale. 96°—10.0, Aq. dest. 90.0). Over-stained sections are differentiated under the control of a microscope in a 2.5% solution of iron alum to the desired degree, washed for ¹/₂–1 hour in running water, and mounted in Canada balsam. Results—black-blue to gray-black shades. Sharp staining of nuclei, centrosomes, cilia, and other cell elements. Additional staining: Eosin, Erythrosin, Rubin S., Chromotrop, Lichtgrun. Staining with all hematoxylins gives excellent results not only on sections, but also on whole mounts of protozoa and free cell elements after wet fixation. However, it must be remembered that iron hematoxylin stains the most diverse inclusions in the cell that are completely dissimilar in their chemical composition and morphological significance, and therefore the data obtained with this staining require very careful evaluation. Prices: Haematoxylin puriss. 10 g—27.70 Mark, Haematoxylin Böhmer 100 cubic cm—1.15 M, Haematoxyl. Delafield 100 cubic cm—1.50 M, Haematoxyl. Ehrlich 100 cubic cm—2.00 M.