Trichrome staining is a

histological Histology, also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissue (biology), tissues. Histology is the microscopic counterpart to gross anatomy, which looks at large ...

staining Staining is a technique used to enhance contrast in samples, generally at the Microscope, microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissue (biology), tissues), in cytology (microscopic ...

method that uses two or more

acid dye An acid dye is a dye that is typically applied to a textile at low pH. They are mainly used to dye wool, not cotton fabrics. Some acid dyes are used as food colorants, and some can be used to stain organelles for medical microscopy. Acid dyes ...

s in conjunction with a polyacid. Staining differentiates tissues by tinting them in contrasting colours. It increases the contrast of microscopic features in cells and tissues, which makes them easier to see when viewed through a

microscope A microscope () is a laboratory equipment, laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic ...

. The word '' trichrome'' means "three colours". The first staining protocol that was described as "trichrome" was Mallory's trichrome stain, which differentially stained

erythrocyte Red blood cells (RBCs), referred to as erythrocytes (, with -''cyte'' translated as 'cell' in modern usage) in academia and medical publishing, also known as red cells, erythroid cells, and rarely haematids, are the most common type of blood ce ...

s to a red colour,

muscle tissue Muscle is a soft tissue, one of the four basic types of animal tissue. There are three types of muscle tissue in vertebrates: skeletal muscle, cardiac muscle, and smooth muscle. Muscle tissue gives skeletal muscles the ability to contract. ...

to a red colour, and

collagen Collagen () is the main structural protein in the extracellular matrix of the connective tissues of many animals. It is the most abundant protein in mammals, making up 25% to 35% of protein content. Amino acids are bound together to form a trip ...

to a blue colour. Some other trichrome staining protocols are the Masson's trichrome stain, Lillie's trichrome, and the

Gömöri trichrome stain Gömöri trichrome stain is a histological stain used on muscle tissue. It can be used to test for certain forms of mitochondrial myopathy Mitochondrial myopathies are types of myopathy, myopathies associated with mitochondrial disease. Adeno ...

Purpose

Without trichrome staining, discerning one feature from another can be extremely difficult.

Smooth muscle tissue Smooth muscle is one of the three major types of vertebrate muscle tissue, the others being skeletal muscle, skeletal and cardiac muscle. It can also be found in invertebrates and is controlled by the autonomic nervous system. It is non-striated ...

, for example, is hard to differentiate from collagen. A trichrome stain can colour the muscle tissue red, and the collagen fibres green or blue. Liver biopsies may have fine collagen fibres between the liver cells, and the amount of collagen may be estimated based on the staining method. Trichrome methods are now used for differentiating muscle from collagen, pituitary alpha cells from beta cells, fibrin from collagen, and

mitochondria A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is us ...

in fresh frozen muscle sections, among other applications. It helps in identifying increases in collagenous tissue (i.e., fibrotic changes) such as in

liver cirrhosis Cirrhosis, also known as liver cirrhosis or hepatic cirrhosis, chronic liver failure or chronic hepatic failure and end-stage liver disease, is a chronic condition of the liver in which the normal functioning tissue, or parenchyma, is replaced ...

and distinguishing tumours arising from muscle cells and fibroblasts.

Procedure

Trichrome staining techniques employ two or more

s. Normally acid dyes would stain the same basic proteins, but by applying them sequentially the staining pattern can be manipulated. A polyacid (such as

phosphomolybdic acid Phosphomolybdic acid is the heteropolymetalate with the formula . It is a yellow solid, although even slightly impure samples have a greenish coloration. It is also known as dodeca molybdophosphoric acid or PMA, is a yellow-green chemical compound ...

Phosphotungstic acid Phosphotungstic acid (PTA) or tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula . It forms hydrates . It is normally isolated as the ''n'' = 24 hydrate but can be desiccated to the hexahydrate (''n'' = 6). EPTA is the na ...

) is used to remove dye selectively. Polyacids are thought to behave as dyes with a high

molecular weight A molecule is a group of two or more atoms that are held together by Force, attractive forces known as chemical bonds; depending on context, the term may or may not include ions that satisfy this criterion. In quantum physics, organic chemi ...

: they displace easily removed dye from collagen. Usually a red dye in dilute

acetic acid Acetic acid , systematically named ethanoic acid , is an acidic, colourless liquid and organic compound with the chemical formula (also written as , , or ). Vinegar is at least 4% acetic acid by volume, making acetic acid the main compone ...

is applied first to overstain all components. Then a polyacid is applied to remove the red dye from collagen and some other components by displacement. A second acid dye (blue or green) in dilute acetic acid is applied which, in turn, displaces the polyacid, resulting in collagen stained in a contrasting colour to the initial dye used. If erythrocytes are to be stained, a small molecular weight yellow or orange dye is applied before staining with the red dye. It is usually applied from a saturated solution in 80% ethanol and often in conjunction with picric acid (itself a dye) and a polyacid. The methods exploit minor differences in tissue reaction to dyes, density, accessibility and so on. Trichrome stains in which dyes and a polyacid are applied sequentially are called ''multi-step trichromes''. In "one-step" methods, all the dyes—with or without a polyacid—are combined in a single solution. One of the oldest single-step approaches to trichrome staining is van Gieson's method, which stains muscle and

cytoplasm The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...

yellow, and collagen red. Another is the

, which closely mimics Masson's trichrome. In "yellowsolve" methods, a red dye in dilute acetic acid is first applied, then the section is very thoroughly dehydrated to ensure that no moisture remains. The red dye is then displaced by a yellow dye in a solvent, such as cellosolve (2-ethoxy-ethanol). The name ''yellowsolve'' is a blend of the terms ''yellow'' and ''cellosolve''. Lendrum's phloxine-tartrazine for cell inclusions is one example of a yellowsolve stain.

Dyes

Among the dyes used for trichrome staining are: ;Red : Acid fuchsin, xylidine ponceau, chromotrope 2R, Biebrich scarlet, ponceau 6R, phloxine ;Blue and green :Light green SF yellowish, Fast Green FCF,

methyl blue Methyl blue is a chemical compound with the molecular formula C37H27N3Na2O9S3. It is used as a stain in histology, and stains collagen blue in tissue sections. It can be used in some differential staining techniques such as Mallory's trichrome ...

, water blue ;Yellow :

Picric acid Picric acid is an organic compound with the formula (O2N)3C6H2OH. Its IUPAC name is 2,4,6-trinitrophenol (TNP). The name "picric" comes from (''pikros''), meaning "bitter", due to its bitter taste. It is one of the most acidic phenols. Like ot ...

orange G Orange G also called C.I. 16230, Acid Orange 10, or orange gelb is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder. Staining Orange G ...

, Martius yellow,

tartrazine Tartrazine is a synthetic lemon yellow azo dye primarily used as a food coloring. It is also known as E number E102, C.I. 19140, FD&C Yellow 5, Yellow 5 Lake, Acid Yellow 23, Food Yellow 4, and trisodium 1-(4-sulfonatophenyl)-4-(4-sulfonato ...

, milling yellow

Notes

References

* Baker J.R. (1958) Principles of biological Microtechnique. London: Methuen. (Also paperback reprint, Franklin Classics Trade Press, USA, 2018. ISBN 978-0353331044.) * Horobin R.W. (1982) Histochemistry. Stuttgart: Gustav Fischer. ISBN 3437107003. * Horobin R.W. (1988) Understanding Histochemistry. Chichester: Ellis Horwood. ISBN 9780470210604. * Kiernan J.A. (2015) Histological and Histochemical Methods, 5th ed. Banbury, UK: Scion. ISBN 9781907904325. * Prento P. (2009) Staining of macromolecules: possible mechanisms and examples. Biotech. Histochem. 84: 139-158. * Puchtler H. Isler H. (1958) The effect of phosphomolybdic acid on the stainability of connective tissues by various dyes. J. Histochem. Cytochem. 6: 265-270. * Reid PE. Iagallo M., Nehr S., Jankunis M., Morrow P. (1993) Mechanism of connective tissue techniques. 1. The effect of dye concentration and staining time on anionic dye procedures. Histochem. J. 25: 821-829.

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