Haematoxylin

  Haematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the wood of the logwood tree. When oxidised it forms haematein, a compound with rich blue-purple colour, and is used, together with a suitable mordant (most commonly Fe(III) or Al(III) salts), to stain cell nuclei prior to examination under a microscope. Structures that stain with haematoxylin are called basophilic.

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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. Aluminum salt lakes are usually colored blue white while ferric salt lakes are colored blue-black.

Aluminum haematoxylin solutions

The two main alum haematoxylin solutions employed are Ehrlich's haematoxylin and Harris haematoxylin. Alum haematoxylin solutions impart on the nucleus a light transparent blue stain which rapidly turns red in the presence of an acid.

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

When tap water is not sufficiently alkaline, or is even acid and is unsatisfactory for blueing haematoxylin, tap water substitute consisting of 33.5 g NaHCO₃ and 20 g MgSO₄ in one liter of water with thymol (to inhibit formation of molds), is used to accelerate blueing of thin paraffin sections. Use of very cold water slows down the process while warming accelerates it. In fact, the use of very cold water below 10 °C for blueing sections may even produce pink artifact discolorations on the tissue.

See also

• staining (biology)
• Histology
• Eosin
• H&E stain
• Haematin