Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree (Haematoxylum campechianum) with a chemical formula of C16H14O6. This naturally derived dye has been used as a histologic stain, as an ink and as a dye in the textile and leather industry. As a dye, haematoxylin has been called palo de Campeche, logwood extract, bluewood and blackwood. In histology, haematoxylin staining is commonly followed by counterstaining with eosin. When paired, this staining procedure is known as H&E staining and is one of the most commonly used combinations in histology. In addition to its use in the H&E stain, haematoxylin is also a component of the Papanicolaou stain (or Pap stain) which is widely used in the study of cytology specimens. Although the stain is commonly called haematoxylin, the active colourant is the oxidized form haematein, which forms strongly coloured complexes with certain metal ions (commonly Fe(III) and Al(III) salts). In its pure form, haematoxylin is a colourless and crystalline solid, although commercial samples are typically light to dark brown based on the level of impurities present.
Extraction and purification
Haematoxylin has been synthesized, although never in commercially viable amounts. Historically the logwood was exported and the haematoxylin extracted in Europe. More recently extraction takes place closer to where the logwood is harvested. Extraction of haematoxylin from logwood on industrial scales has been accomplished in the 'French process' by boiling the wood chips or in the 'American process' with steam and pressure. Once extracted, the dye can be sold as a liquid concentrate or dried and sold in a crystalline form. Modern production methods use water, ether or alcohol as a solvent, at which point the extracts may be further refined to the level of purity needed. The commercial product may vary from batch to batch and between manufacturers in both the level of impurities and in the ratio of haematoxylin to haematein. For histologic use, this variability can affect the stain's interaction with biological tissue samples, and is therefore of concern to histologists and pathologists. Haematoxylin, like other biological stains, may be certified by the Biological Stain Commission, signifying that a particular batch of stain works in a standardized test, although this does not specify the dye's actual purity.
Use as a histologic stain
Haematoxylin stain is commonly followed (or counterstained) with another histologic stain, eosin. When paired, this staining procedure is known as H&E staining, and is one of the most commonly used combinations in histology. Haematoxylin is also a component of the Papanicolaou stain (or PAP stain) which is widely used in the study of cytology specimens, notably in the PAP test used to detect cervical cancer. Principally used as a nuclear stain (to stain the cell nucleus), haematoxylin will also stain rough endoplasmic reticulum, ribosomes, collagen, myelin, elastic fibers, and acid mucins. Haematoxylin alone is not an effective stain, but when oxidized to hematein, and combined with a mordant, stains chromatin in cell nuclei dark blue to black. The colour and specificity of haematoxylin stains are controlled by the chemical nature, and amount, of the mordant used, and the pH of the staining solution, thus, a variety of haematoxylin formulations have been developed.
Stain formulations
Haematoxylin stain formulations can be broadly classified based on how the haematoxylin is oxidized (or ripened) and by choice of the mordant used. Haematoxylin stain formulations may either be natural oxidized by exposure to air and sunlight, or more commonly, especially in commercially prepared solutions, chemically oxidized using sodium iodate. Commonly only enough oxidizer is added to convert one half of the haematoxylin to haematein, allowing the remainder to naturally oxidize during use, this extends the staining solution's useful life as more haematein is produced, while some haematein is further oxidized to oxyhaematein. Of the metallic salts used as mordants, aluminium is the most common, other mordants include salts of iron, tungsten, molybdenum and lead. Depending on the formulation or staining technique, haematoxylin stains may be used in what is called a progressive manner, in which the length of time the tissue remains in contact with the staining solution is used to control the amount of colouration, or in a regressive manner, in which the tissue is over-stained, and excess stain is removed in a secondary step of the procedure. Removal of unwanted staining, or differentiation, typically involves a solution of diluted ethanol and hydrochloric acid.
Table of significant formulations
Early use as a histologic stain In 1758, Georg Christian Reichel used haemotoxylin, without a mordant, to stain plant tissues. John Thomas Quekett in an 1852 book, suggests using "logwood" (haematoxylin) to dye translucent material for examination under the microscope. In 1863, Wilhelm von Waldeyer-Hartz used haematoxylin on animal tissue without a mordant (with limited success), and is sometimes credited as being the first to do so, although this is not universally accepted. Franz Böhmer in 1865 published a haematoxylin formula using alum as a mordant, and in 1891, Paul Mayer published a formulation using a chemical oxidizer to convert haematoxylin into haematein. The first use of haematoxylin with eosin as a counterstain, which is currently the most used stain combination in histology, was first suggested by A. Wissowzky in 1876. By the early 1900s, haematoxylin had become widely accepted as a histologic stain.
… excerpt ends here. Continue reading the full article.
