Hematoxylin & Hematein
Hematoxylin is a natural dye which is extracted from the heartwood of the tree Haematoxylum campechianum, although histotechnologists are probably more familiar with the name as Hematoxylon campechianum. The genus name Hematoxylum is derived from two Greek words: haimatos which means blood, and xylon which means wood.
There is an interesting article on hematoxylin and other logwoods at Wayne's Word. The article describes how logwood dyes were introduced to Europe, and how they resulted in the creation of two countries.
|Leaves and flowers of H. campechianum courtesy of James Cook University, Queensland, Australia.|
There is a photograph of a mature tree at Dave's Garden
The hematoxylin which we buy is extracted from the heartwood of this bloodwood tree. There may be some differences in method, but one is to chip the heartwood of freshly logged trees, then boil the chips in water. An orange-red solution is obtained, which turns yellow, then black on cooling. The water is evaporated leaving crude hamatoxylin. Depending on the genetic line of the tree, hematoxylin content ranges from 0% - 10%. Further purification is undoubtedly done.
Although it is common practice to use hematoxylin, it is not itself the dye. During the preparation of staining solutions hematoxylin is converted into hematein. This is usually accomplished with chemical oxidising agents, but is sometimes accomplished by atmospheric oxygen over time. Sodium iodate is the most commonly used oxidising agent for this purpose (0.2g will oxidise 1g hematoxylin). Others are mercuric oxide (now strongly deprecated because it is poisonous), potassium permanganate and iodine. Hematein may also be referred to as hematoxein, although it is not usually seen in histotechnology references. Hematein is not incorporated directly into most staining solutions because it continues to oxidise in solution and forms non-staining products. The quality of hematoxylin is usually higher and more consistent than the quality of hematein, and solutions made with it are more easily standardised.
The dye is usually used in conjunction with a mordant, the two commonest being aluminum (as ammonium or potassium alum), or iron (ferric chloride or iron alum). Other mordants are used much less frequently but include chrome alum and phosphotungstic acid. The tissue component most frequently demonstrated is nuclear chromatin using an aluminum mordant in the Hematoxylin and Eosin general oversight staining method. Using ferric salts as the mordant, it is also used for acid resistant nuclear staining, the demonstration of muscle striations and numerous other elements. With phosphotungstic acid it can demonstrate fibrin, muscle striations and some neuroglia fibres. There are many published formulas.
Due to the widespread use of this dye in medical histology, it is important that a steady supply be available. This has not always been the case. A shortage occurred in the early part of the 1970s. During this period several dyes were tested as substitutes with some success. Unfortunately, none of them have the wide variety of uses that hematoxylin has. Celestine blue B and mordant blue 3 are probably the most successful.
Hematoxylin has not yet been fully synthesised, but the compound has been split into some precursors, which have been successfully re-converted to the original compound.
Horobin R W & Kiernan J A, (2002)
Conn's Biological Stains, 10th ed.
BIOS Scientific Publishers, Oxford, UK
Susan Budavari, Editor, (1996)
The Merck Index, Ed. 12
Merck & Co., Inc., Whitehouse Station, NJ, USA
R. H. M. J. Lemmens and N. Wulijarna-Soetjipto, Editors. (1992)
Plant resources of South East Asia No. 3, Dye and tannin-producing plants
PROSEA, Bogor, Indonesia.
Morsingh, F., and Robinson, R., (1970)
The synthesis of brazilin and haematoxylin
Tetrahedron, v. 26, p. 281,
James Cook University, School of Marine and Tropical Biology
Townsville, Queensland, Australia