Amyloid Metachromasia

Metachromasia is the colouring of a tissue constituent in a colour different from that of the dye applied. Commonly, this is purple to red shades from application of a blue dye such as toluidine blue, or yellow shades from application of a red dye such as neutral red. This type of metachromasia is due to the presence of acid mucopolysaccharides, particularly if sulphated, so it is often used to detect mucinous substances as these are often mixtures containing different compounds, including some that are strongly sulphated.

Although methyl violets, of which crystal violet is one, do give metachromatic staining, they are not considered to be the most effective for the purpose. The azures or toluidine blue are more effective usually. The exception is for amyloid, when significant metachromasia is given by amyloid deposits using crystal or methyl violets, dyes that would not normally be the first choice to demonstrate metachromasia. In addition, Hoffman's violet (primulin or dahlia) and methyl green may be used.

It was, for a long time, thought that amyloid metachromatic staining was due to preferential staining from a dye mixture of related dyes. That is, that crystal violet, being a mixture of hexamethylpararosaniline containing some less methylated products which were redder in shade, preferentially stained the amyloid with these redder homologues and the background by the crystal violet. It is now clear this is not the case.

Amyloid contains some acid mucopolysaccharides, but its metachromasia with crystal violet does not appear to be primarily due to that. Experiments comparing metachromasia with toluidine blue to that of crystal violet have shown that crystal violet metachromasia is obtained at much lower pH levels than toluidine blue metachromasia, indicating a clear difference between the two. The metachromasia of amyloid with crystal violet appears to be based on hydrogen bonding and the regular alignment of dye molecules.

The metachromatic colour change is visually quite distinct: deep red-purple on a blue background. It is often brighter with an aqueous mountant but may leach from the sections with a sugary mounting medium, i.e. staining may be transient. As with other metachromatic stains, clearing the section by repeatedly blotting then flooding with xylene may enable a resinous mounting medium to be used, albeit with somewhat reduced intensity of metachromatic staining.

It is sometimes stated that methods for crystal violet metachromasia of amyloid are now obsolete, since other dye staining methods are easily available. The problem, repeating it once again, is that methods for amyloid are not completely reliable since amyloid itself is so variable, and sometimes these other methods fail to stain adequately. On those occasions crystal violet metachromasia may prove very useful. However, as with all methods for amyloid, it should be noted that some amyloid may also fail to stain with this technique.

Crystal violet is a degradation product of methyl green, so this dye has also been used for metachromatic amyloid staining and gives purple-red amyloid on a green background, thus increasing contrast. For this purpose it is important that the methyl green contain a significant amount of crystal violet. This is the opposite of what is needed for staining nucleic acids with Trevan and Sharrock's variant of the Unna Pappenheim method, which employs methyl green and pyronin, and in which the methyl green should be as free from crystal violet as possible, even to the extent of removing any excess by washing the solution with chloroform. Perhaps different samples of methyl green should be kept for the two purposes, if necessary, or the methyl green solution for amyloid staining should have enough crystal violet added to bring about distinct metachromatic staining. It is important to note that purified methyl green which contains no crystal violet will not be suitable for metachromatic amyloid staining.

For amyloid staining, crystal violet is often preferred to methyl violet as the orthochromatic shade is a little bluer and the purple-red of the amyloid contrasts more effectively with this than with the slightly redder methyl violet. Neither the plain crystal/methyl violet nor the methyl green methods are difficult to use and the procedures are quite straightforward. They involve staining a frozen or dewaxed and hydrated paraffin section with an aqueous solution of the dye for a few minutes, then washing off the dye and removing any excess with a dilute acetic acid solution.

Metachromatic Staining Methods
Bancroft methyl green
Bancroft crystal violet
Birch-Hirschfeld crystal violet
Highman crystal violet
Jurgens crystal violet
Lendrum crystal violet

 

Reference
Carnes, W H, & Forker, B R, (1956),
Metachromasy of amyloid: a spectrophotometric study with particular reference to the dye chromotrope bond.
Lab. Investigation, v 5, page 21.

 


 

 

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