Metachromasia (var. metachromasy) is a characteristical change in the

color Color (American English) or colour (British English) is the visual perceptual property deriving from the spectrum of light interacting with the photoreceptor cells of the eyes. Color categories and physical specifications of color are associ ...

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 ...

carried out in biological tissues, exhibited by certain

dye A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...

s when they bind to particular substances present in these tissues, called chromotropes. For example,

toluidine blue Toluidine blue, also known as TBO or tolonium chloride (International Nonproprietary Name, INN) is a blue cationic (basic) dye used in histology (as the toluidine blue stain) and sometimes clinically. Test for lignin Toluidine blue Solution (ch ...

becomes dark blue (with a colour range from blue-red dependent on glycosaminoglycan content) when bound to

cartilage Cartilage is a resilient and smooth type of connective tissue. In tetrapods, it covers and protects the ends of long bones at the joints as articular cartilage, and is a structural component of many body parts including the rib cage, the neck an ...

. Other widely used metachromatic stains are the haematological Giemsa and May-Grunwald stains that also contain thiazine dyes. The white cell nucleus stains purple, basophil granules intense magenta, whilst the cytoplasms (of mononuclear cells) stains blue. The absence of color change in staining is named

orthochromasia In chemistry, orthochromasia is the property of a dye or stain to not change color on binding to a target, as opposed to metachromatic stains, which change color. The word is derived from the Greek '' orthos'' (correct, upright), and chromatic ( ...

. The underlying mechanism for metachromasia requires the presence of

polyanions 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 ...

within the tissue. When these tissues are stained with a concentrated basic dye solution, such as toluidine blue, the bound dye molecules are close enough to form dimeric and polymeric aggregates. The light absorption spectra of these stacked dye aggregates differ from those of the individual monomeric dye molecules. Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups—such as the ground substance of cartilage, heparin-containing granules of mast cells, and rough endoplasmic reticulum of plasma cells—exhibit metachromasia. This depends on the charge density of the negative sulfate and carboxylate anions in the glycosaminoglycan (GAG). The GAG polyanion stabilizes the stacked, positively-charged dye molecules, resulting in a spectral shift as the conjugated double bond π-orbitals of adjacent dye molecules overlap. The greater the degree of stacking, the greater the metachromatic shift. Thus, hyaluronic acid, lacking sulphate groups and with only moderate charge density, causes slight metachromasia; chondroitin sulfate, with an additional sulfate residue per GAG saccharide dimer, is an effective metachromatic substrate, whilst heparin, with further N-sulfation, is strongly metachromatic. Therefore, toluidine blue will appear purple to red when it stains these components. The metachromatic properties of dimethylmethylene blue, a thiazine dye closely related to toluidine blue, have been exploited to assay glycosaminoglycans extracted from cartilage and other connective tissues. The absorption peak shifts from about 630 nm (red absorption, therefore blue colour) to about 530 nm in the presence of GAG. Humbel and Etringer's original assay was developed by others to create a stable and widely used dimethylmethylene blue reagent. Although metachromasia was observed and described since 1875, by

Cornil Cornil (; oc, Cornilh) is a commune in the Corrèze department in central France. Cornil station has rail connections to Brive-la-Gaillarde, Ussel, Tulle and Bordeaux. Population See also *Communes of the Corrèze department The followin ...

, Ranvier and others, it was the German scientist

Paul Ehrlich Paul Ehrlich (; 14 March 1854 – 20 August 1915) was a Nobel Prize-winning German physician and scientist who worked in the fields of hematology, immunology, and antimicrobial chemotherapy. Among his foremost achievements were finding a cure ...

(1854-1915) who gave its name and studied it more extensively. The modern understanding of metachromasia was advanced by Belgian histologist

Lucien Lison Lucien Alphonse Joseph Lison (1908–1984) was a Belgian/Brazilian physician and biomedical scientist, considered the "father of histochemistry".Ronan O’RahillyThree and one-half centuries of histology ''Irish Journal of Medical Science (19 ...

, who studied it between 1933 and 1936 and ascertained its value in the quantitative determination of sulfate

ester In chemistry, an ester is a compound derived from an oxoacid (organic or inorganic) in which at least one hydroxyl group () is replaced by an alkoxy group (), as in the substitution reaction of a carboxylic acid and an alcohol. Glycerides ar ...

s of 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 ...

. He also studied the metachromasia of

nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...

s. More recently, Karlheinz Toepfer published in 1970 spectral shifts with increasing concentration of the thiazine dyes that matched the spectra of dye:heparin mixtures, showing clearly that metachromasia, corresponding to the colour of stained cartilage, could be reproduced by high concentration of the dye alone in solution. Hence, proximity of the dye molecules was the key parameter in defining metachromasia. Another example of metachromatic dye (fluorochrome) is acridine orange. Under certain conditions it stains single-stranded nucleic acids fluorescing red (red luminescence) while when interacts with double stranded nucleic acids gives green fluorescence. Darzynkiewicz Z, Kapuscinski J. (1990)“Acridine Orange, a Versatile Probe of Nucleic Acids and Other Cell Constituents.” Chapter in: Flow Cytometry and Sorting. Melamed MR, Mendelsohn M & Lindmo T (eds), Alan R. Liss, Inc., New York. pp. 291-314. ISNBM 0-471-56235-1

References

Further reading