Metachromasia

Metachromasia is a phenomenon not infrequently encountered in dye staining. It is often seen following staining with a solution of a simple blue dye such as methylene blue or toluidine blue, particularly if the preparation is examined mounted with water, although those dyes are not the only ones that give this effect. Generally the tissue components will be stained blue by the blue dye, but some tissue components will stain red. The usual colour of the dye is termed the orthochromatic colour and the altered colour is the metachromatic colour. The substance stained metachromatically is termed a chromotrope.

Metachromasia may be defined as:

The staining of tissue elements in two colours from a solution of a single dye.

The phrase "a solution of a single dye" in this context means a solution of a single chemical compound. In other words, the two colours obtained are produced by the single chemical compound (the single dye) attaching to the tissues in different fashions, presumably. This definition therefore eliminates mixtures of dyes as the source of the two colours. Thus, adding phloxine to a solution of eosin in order to stain some elements in a redder shade than the pink of eosin is not an example of metachromasia, rather it is an example of selective or preferential staining. The dyes used to display metachromasia are basic dyes. An equivalent phenomenon with acid dyes does not exist.

As it happens, metachromasia is often seen with solutions of methylene blue, especially old solutions that have been made for some time. Fresh solutions of this dye may, or may not, show metachromasia depending on the purity of the dye. Over some months the methylene blue will develop the ability to show distinct metachromasia, at which point it is referred to as polychrome methylene blue. This change in staining comes about due to the methylene blue molecule converting to other, related, compounds, such as azure A, azure B and azure C, a process often referred to as "ripening". In other words, the solution of methylene blue becomes a mixture of related dyes. How then is it still to be considered an example of metachromasia if it is not a solution of a single chemical compound? The answer to that question lies in the fact that if the oxidation products of methylene blue, i.e. any one of the dyes mentioned, is isolated and purified and then dissolved and used for staining appropriate tissue, they would exhibit metachromasia. So while the methylene blue solution is a mixture of related dyes, the metachromasia itself is due to each of the dyes independently staining in two colours. These dyes are all blue, yet the colours produced by them are blue and red. However, none of the dyes are red in solution. The red colour is produced from the blue dyes interacting with the tissue. This is different from the example above of eosin and phloxine, where each of the two dyes preferentially stain different tissue components in their usual colour whereas each of the methylene blue homologues stains in two different colours from a single chemical compound.

The most common dyes used to stain metachromatically give blue orthochromatic staining with some elements metachromatically stained red. However, this is by no means the only colour combination. Both safranin O and neutral red are metachromatic dyes which have red orthochromatic staining and yellow metachromatic staining. In fact, for many years in the early 20th century, safranin O was used to demonstrate cartilage metachromatically yellow in contrast to red nuclei and a pink background.

At some point it was observed that metachromatically stained tissues were acid mucopolysaccharides, and it is now known that these are the commonest tissue components that are stained. The basic metachromatic dyes attach to available acid groups on mucopolysaccharide molecules, both carboxyl and sulphate. Consequently, the vast majority of acid mucopolysaccharides are metachromatic, but sulphated acid mucopolysaccharides are intensely so. Since most "mucin" exists as mixtures of different mucopolysaccharides, both neutral and acid, including sulphated types, metachromasia came to be seen as a standard method for its demonstration. That there must be some acid mucopolysaccharide present explains why metachromatic staining of mucin may sometimes fail. This would be in cases where there is only neutral mucopolysaccharide present. Keep in mind, though, that just as not all mucin is acid mucopolysaccharide, so not all acid mucopolysaccharide is mucin. For instance, mast cells contain the sulphated acid mucopolysaccharide heparin and this is strongly metachromatic, forming the basis for many staining methods.

The colour shift is usually blue to red or red to yellow. That is, the shift is to shorter wavelengths of light. This is termed a hypsochromic shift in absorption. The converse, a shift to longer wavelengths is know as a bathochromic shift, but is not usually encountered in most histological applications. Baker does mention an example with toluidine blue, but it is an obscure application.

It should be noted at this point that amyloid may stain metachromatically withmethyl violet and its homologues. Amyloid is not generally thought to be an acid mucopolysaccharide and the basis for amyloid's metachromasia is different from that of acid mucopolysaccharides.

When staining with toluidine blue, you may encounter references to α-metachromasia (alpha metachromasia), β-metachromasia (beta metachromasia) and γ-metachromasia (gamma metachromasia). These terms are sometimes used to distinguish between non-metachromatic staining (α-metachromasia), strongly positive red staining (γ-metachromasia), and weakly positive violet or purple staining, i.e. intermediate between blue and red (β-metachromasia). They are considered to give an indication as to whether the staining is in the form of a dye monomer (α-metachromasia), a short dye polymer of two or three or so (β-metachromasia) or a longer dye polymer (γ-metachromasia). At best this should be considered indicative only, not an absolute.

Metachromasia of acid mucopolysaccharides
The metachromatic staining of acid mucopolysaccharides appears to be due to the dye attaching to the metachromatic substance in a form which incorporates a water molecule linking adjacent dye molecules in sequence and forming long chains, whereas orthochromatically stained materials have a dye molecule discretely attached. This linked sequence of molecules has different light absorption characteristics, presumably due to changes in the energy of the delocalised electron clouds brought about by extending the dye molecule with water molecules, so that it appears in a different colour to the orthochromatic colour.

It is well known that dehydrating metachromatic preparations reduces the intensity of the metachromasia and may destroy it altogether. Instructions for dehydration techniques which are claimed to preserve metachromatic staining nearly always involve reducing the effectiveness of the dehydration step, perhaps by blotting sections to avoid any alcohol immersion step, drying the section by waving in air, substituting a less effective dehydrant such as tertiary butanol, or passing through ethanol very rapidly. However it is done, the results usually give an inferior preparation with a notably reduced intensity of metachromasia. If metachromatic staining is used in critical applications, the preparation should be examined mounted in distilled water. It is also sometimes recommended that the blue, daylight filter of the microscope be removed so that the light is a little warmer to accentuate the colour shift.

Metachromasia of amyloid
Amyloid is predominantly formed from proteins with a β-pleated sheet structure. It may also contain some carbohydrates, is variably positive with PAS and may stain with dyes, such as alcian blue, which are usually used for acid mucopolysaccharides. Amyloid metachromasia is seen with methyl violet and its homologues. It is believed that this dye attaches to the amyloid in a regular pattern forming what is sometimes referred to as a "pseudocrystal", and it is the regular alignment of monomeric dye molecules which causes the colour shift rather than the polymerisation produced when a basic, positively charged dye attaches to regularly spaced, negatively charged tissue groups which are so important in acid mucopolysaccharide metachromasia. Dehydration with ethanol reduces or eliminates the metachromatic effect, but blotting a section dry usually retains sufficient for the preparation to be useful.

Metachromatic dyes
DyeCI #OrthochromaticMetachromaticClass
Methylene blue52015BlueRedThiazin
Azure A52005BlueRedThiazin
Azure B52010BlueRedThiazin
Azure C52002BlueRedThiazin
Thionin52000BlueRedThiazin
Toluidine blue52040BlueRedThiazin
Neutral red50040RedYellowEurhodin
Safranin O50240RedYellowSafranin
Bismarck brown Y21000BrownYellowAzo
Methyl violet42535VioletRedTriarylmethane

 

Reference
Wikipedia has an article on Metachromasia

Baker, John R., (1958)
Principles of biological microtechnique
Methuen, London, UK.

Drury, R A, and Wallington, E A, (1967).
Carleton's histological technique., Ed. 5.
Oxford University Press, London, England.

Culling, C F A, Allison, R T, Barr, W T, (1985).
Cellular pathology technique., Ed. 4.
Butterworths, London, England.

 


 

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