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IUPAC name
CAS number 517-28-2
PubChem 10603
MeSH Hematoxylin
Molecular formula C16H14O6
Molar mass 302.28 g mol−1
Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)
Infobox references

Haematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the wood of the logwood tree. When oxidized it forms haematein, a compound that forms strongly colored complexes with certain metal ions, notably Fe(III) and Al(III) salts. Metal-haematein complexes are used to stain cell nuclei prior to examination under a microscope. Structures that stain with iron- or aluminium-haematein are often called basophilic, even though the mechanism of the staining is different from that of staining with basic dyes.

Its CAS number is [517-28-2] and its SMILES structure is OC(C(O)=C4)=C C1=C4CC3(O) C1C2=CC=C(O) C(O)=C2OC3.

Haematoxylin and eosin stain is one of the most commonly used stains in histology. It is a permanent stain as opposed to temporary stains (e.g. iodine solution in KI).

Other common stain is phosphotungstic acid haematoxylin, a mix of haematoxylin with phosphotungstic acid.

In 1970s, due to clear felling of forests in Brazil and Central America, there was a shortage of logwood and therefore of haematoxylin. Its price went to record heights, which affected the cost of diagnostic histopathology, and prompted a search for alternative nuclear stains. Before the use of any alternatives became firmly established, haematoxylin returned to the market, though at a higher price, and resumed its place in histopathology. There were several dyes recommended as replacements: Celestine blue B (CI 51050), Gallocyanin (CI 51030), Gallein (CI 45445) and Solochrome cyanin (CI 43820). All four used Fe(III) as the mordant. Another alternative is the red dye brazilin, which differs from haematoxylin by only one hydroxyl group.


Haematoxylin staining solutions

These stains are commonly employed for histologic studies. The mordants used to demonstrate nuclear and cytoplasmic structures are alum and iron, forming lakes or colored complexes (dye-mordant-tissue complexes), the color of which will depend on the salt used. Aluminium salt lakes are usually colored blue white while ferric salt lakes are colored blue-black.


Aluminium haematoxylin solutions

The three main alum haematoxylin solutions employed are Ehrlich's haematoxylin, Harris's haematoxylin and Mayer's haematoxylin. The name haemalum is preferable to "haematoxylin" for these solutions because haematein, a product of oxidation of haematoxylin, is the compound that combines with aluminium ions to form the active dye-metal complex. Alum haematoxylin solutions impart to the nuclei of cells a light transparent red stain which rapidly turns blue on exposure to any neutral or alkaline liquid.

Alum or potassium aluminium sulfate used as the mordant usually dissociates in an alkaline solution, combining with OH of water to form insoluble aluminium hydroxide. In the presence of excess acid, aluminium hydroxide cannot be formed thus failure of aluminium haematoxylin dye-lake to form, due to lack of OH ions. Hence, acid solutions of alum haematoxylin become red. During staining alum haematoxylin stained sections are usually passed on to a neutral or alkaline solution (e.g. hard tap water or 1% ammonium hydroxide) in order to neutralize the acid and form an insoluble blue aluminium haematin complex. This procedure is known as blueing.

When tap water is not sufficiently alkaline, or is even acid and is unsatisfactory for blueing haematoxylin, a tap water substitute consisting of 33.5 g NaHCO3 and 20 g MgSO4.7H2O in one litre of water with thymol (to inhibit formation of moulds), is used to accelerate blueing of thin paraffin sections. Addition of a trace of any alkali to tap or distilled water also provides an effective blueing solution; a few drops of strong ammonium hydroxide or of saturated aqueous lithium carbonate, added immediately before use, are sufficient for a 400 ml staining dish full of water. Use of very cold water slows down the blueing process, whereas warming accelerates it. In fact, the use of water below 10°C for blueing sections may even produce pink artifact discolorations in the tissue.

See also

External links


Brown, G. G. 1978. An Introduction to Histotechnology. Appleton-Century-Crofts, New York.

Jocelyn H. Bruce-Gregorios, M.D.: Histopathologic Techniques, JMC Press Inc., Quezon City, Philippines, 1974.

Meloan, S. M. & Puchtler, H. 1987. "Harris hematoxylin," what Harris really wrote and the mechanism of hemalum stains. Journal of Histotechnology 10: 257-261.

Puchtler, H., Meloan, S.N. & Waldrop, F.S. 1986. Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353-364.


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