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 color, 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.
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.
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 an alkaline solution (e.g. 1% hydroxide) in order to neutralize the acid and free the OH group, to form an insoluble blue aluminium 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.
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