Dye Origins Hematoxylin is extracted from the logwood tree and purified. It is then oxidized and combined with a mordant (typically aluminum) to allow it to bind to the cell structures. Of the many hematoxylin preparations used in histology Gill’s hematoxylin, Harris's hematoxylin and Mayer's hematoxylin are the most popular. Eosin is formed by a reaction between bromine and fluorescein. There are two eosin variants typically used in histology: eosin Y which is slightly yellowish and eosin B which is slightly bluish. Eosin Y is most popular. Special Stains The term special stains traditionally referred to any staining other than an H&E. It covers a wide variety of methods that may be used to visualize particular tissue structures, elements, or even microorganisms not identified by H&E staining. Other methods of staining use immunohistochemistry or in situ hybridization to target specific proteins or DNA/RNA sequences. These methods were sometimes also included as members of the “special stains” family. However, they are quite different in method and purpose and are now typically separated into a third category known as “advanced stains”. While there are literally hundreds of special stains for all manner of purposes, only a few are used with any regularity in clinical histology. The variety of stains also means that special staining is not as automated as H&E staining. While many larger laboratories do use automated instruments for the more common stains, they still have an area for hand staining. The complexity of some stains also works against the uses of automation. Some Common Special Stains. The images below illustrate some of the common special stains and their applications. Masson's Trichrome (skin). This stain is intended for use in histological observation of collagenous connective tissue fibers in tissue specimens. It is used to assist in differentiating collagen and smooth muscle in tumors and assists in the detection of diseases or changes in connective/muscle tissue. Modified GMS Silver Stain (Left: Pneumocystis, lung) (Right: Aspergillus infection, lung). The Modified GMS Silver stain is intended for use in histological observation of fungi, basement membrane and some opportunistic organisms such as pneumocystis carinii in tissue specimens. Periodic Acid Schiff P.A.S (kidney). PAS staining is mainly used for staining structures containing a high proportion of carbohydrates such as glycogen, glycoproteins, proteoglycans typically found in connective tissues, mucus and basement membranes. Often used to stain kidney biopsies, liver biopsies, certain glycogen storage diseases in striated muscles and suspected fungal infections. Perls’ Prussian Blue Iron (liver). This stain is used to detect and identify ferric (Fe3+) iron in tissue preparations, blood smears, or bone marrow smears. Minute amounts of ferric iron (haemosiderin) are commonly found in bone marrow and in the spleen. Abnormal amounts of iron can indicate hemochromatosis and hemosiderosis. Ziehl Neelsen (Acid Fast Bacillus, lung). This stain is used to detect and identify acid fast bacilli in tissue. Bacilli are rod-shaped bacterial organisms. A primary function of this stain is to identify tuberculosis in lung tissue. Alcian Blue (intestine). Alcian Blue is normally prepared at pH 2.5 and is used to identify acid mucopolysaccharides and acidic mucins. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis. Alcian Blue and PAS (intestine). A stain that combines the properties of both Alcian Blue and Periodic Acid Schiff staining. Gomori Trichrome (blue) (submucosa). Trichrome stains are used to stain and identify muscle fibers, collagen and nuclei. They can be used to contrast skeletal, cardiac or smooth muscle. The Gomori Trichrome is a simplification of the more elaborate Masson trichrome stain and combine the plasma stain (chromotrope 2R) and connective tissue stain to provide a brilliant contrasting picture. Gomori Trichrome (green) (submucosa). Trichrome stains are used to stain and identify muscle fibers, collagen and nuclei. They can be used to contrast skeletal,cardiac or smooth muscle. The Gomori Trichrome is a simplification of the more elaborate Masson stain and combines the plasma stain (chromotrope 2R) with the connective tissue stain to provide a brilliant contrasting picture. In the modern age of histology there have been significant improvements in histological stains and techniques. Advanced histological techniques are immunohistochemistry, antibody binding and electron microscopy. In the same line, advanced stains include: Immunohistochemical (IHC), routine hematoxylin eosin (H&E) and the in situ hybridization. Modern stains used are; Masson's Stain used in connective tissues Golgi Stain used in neuronal fibers Toluidine Blue. Verhoeff van Gieson. Perussian Blue iron. Immunological labeling that have fluorescent or enzymatic stains Kluver-Barrera Stain used in Lipofuscin Periodic Acid-Schiff (PAS) Stain used in carbohydrates. Bouin’s fluid. Methyl blue. Methyl red. Others stains are: - Carmine It is a commonly used stain in histology. The stain was used to study microscopic tissue structures when in ammoniacal solution form and it is still used today in histologic studies. Silver Nitrate Silver Nitrate has had a long usage in historical staining techniques and is still used in modern pathology. The Hematoxylin and Eosin Procedures Although historically used, there have been great laboratory changes in Hematoxylin stains; nearly all tissue specimens are treated with Hematoxylin and Eosin today. Romanowsky Stains–Giemsa Stains They were developed in the 1891 by Dimitri Romanowsky and popular for its multicolor in identifying blood parasites. The Giemsa Stains procedure is still used today. There has been great improvement in the stains, and its various methods make it applicable in paraffin-embedded, formalin-fixed and bone marrow biopsies. Gram Stain The Gram staining method was named after a Danish inventor Hans Christian Gram, who invented it as an approach to differentiating bacteria species in 1875. Gram devised the technique of staining for the purpose of distinguishing the type of bacterium infection and also as a way of making the bacteria visible on selected and stained lung tissues during examination. In a recent case in Kuwait, the Gram staining technique was particularly effective in the diagnosis of Gonorrhea giving 99.4% effective result. The method is still used today especially with paraffin sections and has been modified to suit different substances. Mallory Trichrome Stains Used to reveal different macromolecules that make up the cell. It uses the three stains: aniline blue, acid fuchsin and orange G. As a result, this staining technique can reveal collagen, ordinary cytoplasm and red blood cells. Masson Trichrome stain is used to visualize connective tissues, particularly collagen, in tissue sections. In standard Masson’s trichrome stain collagen is stained blue, nuclei are stained dark brown, muscle tissue is stained red and cytoplasm is stained pink. It comprises of three colours by Claude L. Pierre Masson, mostly it produces red keratin and muscle fibres, blue or green collagen and bone, light red or pink cytoplasm, cell nuclei black to dark brown. Samples to be stained are immersed into the following stains to produce Masson’s trichrome - Wiegert’s hematoxylin, which contains ferric chloride in diluted HCL, hematoxylin in 95% ethanol and potassium ferricyanide solution alkalized by sodium borate. This is used to stain the nuclei. - Plasma stain which contains xylidine ponceau, acid fuchsin, glacial acetic acid and distilled water. Other red acid dyes can be used e.g., Biebrich scarlet in Lillie’s trichrome. - Phosphomolybdic acid in distilled water. - Fiber stain which contains light green SF yellowish, or fast green FCF. Used to stain collagen. If blue is preferred to stain green, methyl blue or water blue can be substituted. .