Trichrome staining is a

histological Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...

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

method that uses two or more

acid dye Acid dyes are anionic, soluble in water and are essentially applied from acidic bath. These dyes possess acidic groups, such as SO3H and COOH and are applied on wool, silk and nylon when ionic bond is established between protonated –NH2 group of ...

s in conjunction with a

polyacid Polyelectrolytes are polymers whose repeating units bear an electrolyte group. Polycations and polyanions are polyelectrolytes. These groups dissociate in aqueous solutions (water), making the polymers charged. Polyelectrolyte properties are th ...

. 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 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 means being invisibl ...

. The word '' trichrome'' means "three colours". The first staining protocol that was described as "trichrome" was

Mallory's trichrome stain Mallory's trichrome stain also called Mallory's Triple Stain is a stain utilized in histology to aid in revealing different macromolecules that make up the cell. It uses the three stains: aniline blue, acid fuchsin, and orange G. As a result, this ...

, which differentially stained

erythrocyte Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

s to a red colour,

muscle tissue Muscle tissue (or muscular tissue) is soft tissue that makes up the different types of muscles in most animals, and give the ability of muscles to contract. Muscle tissue is formed during embryonic development, in a process known as myogenesis. Mu ...

to a red colour, and

collagen Collagen () is the main structural protein in the extracellular matrix found in the body's various connective tissues. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole ...

to a blue colour. Some other trichrome staining protocols are the

Masson's trichrome stain Masson's trichrome is a three-colour staining procedure used in histology. The recipes evolved from Claude L. Pierre Masson's (1880–1959) original formulation have different specific applications, but all are suited for distinguishing cells f ...

, 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. It is named for George Gömöri George Gomori may refer to: * György Gömöri (1904–1957), Hungari ...

Purpose

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

Smooth muscle tissue Smooth muscle is an involuntary non-striated muscle, so-called because it has no sarcomeres and therefore no striations (''bands'' or ''stripes''). It is divided into two subgroups, single-unit and multiunit smooth muscle. Within single-unit mus ...

, 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 Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...

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, and end-stage liver disease, is the impaired liver function caused by the formation of scar tissue known as fibrosis due to damage caused by liver disease. Damage causes tissue repai ...

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 ...

or tungstophosphoric acid) 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 held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...

: 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 component ...

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 In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...

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 Acid fuchsin or fuchsine acid, (also called Acid Violet 19 and C.I. 42685) is an acidic magenta dye with the chemical formula C20H17N3Na2O9S3. It is a sodium sulfonate derivative of fuchsine. Acid fuchsin has wide use in histology, and is one o ...

, xylidine ponceau, chromotrope 2R,

Biebrich scarlet Biebrich scarlet (C.I. 26905) is a molecule used in Lillie's trichrome. The dye was created in 1878 by the German chemist Rudolf Nietzki. Biebrich scarlet dyes are used to color hydrophobic materials like fats and oils. The dye is an illegal dye ...

, ponceau 6R,

phloxine Phloxine B (commonly known simply as phloxine) is a water-soluble red dye used for coloring drugs and cosmetics in the United States and coloring food in Japan. It is derived from fluorescein, but differs by the presence of four bromine atoms at ...

;Blue and green :Light green SF yellowish,

Fast Green FCF Fast Green FCF, also called Food green 3, FD&C Green No. 3, Green 1724, Solid Green FCF, and C.I. 42053, is a turquoise triarylmethane food dye. Its E number is E143. Fast Green FCF is recommended as a replacement of Light Green SF yellowish in ...

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 connect ...

water blue Water blue, also known as aniline blue, Acid blue 22, Soluble Blue 3M, Marine Blue V, or C.I. 42755, is a chemical compound used as a stain in histology. Water blue stains collagen blue in tissue sections. It is soluble in water and slightly ...

;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 el, πικρός (''pikros''), meaning "bitter", due to its bitter taste. It is one of the most acidic ...

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-sulfonatop ...

, 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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