What is Schiff's reagent?

Schiff's reagent is a solution that will combine chemically with aldehydes to form a bright red product. Strictly speaking, ketones also react but for all practical purposes they can be ignored. Many tissue components can be stained this way. Schiff's reagent is made from pararosanilin treated with sulphurous acid. This causes a disruption of the chromophore by the addition of a sulphonic acid group to the central carbon. The sulphurous acid is produced by dissolving sulphur dioxide in water.

In some, usually older, texts you will find Schiff's reagent referred to as leucofuchsin. The prefix leuco means white and refers to the loss of colour in the solution. However, a leucobase proper is produced by reduction, and its colour is restored by oxidation. Schiff's reagent does not have the original pararosanilin colour restored. Rather, a new coloured compound is produced by the chemical combination with an aldehyde. For that reason it is usual now to refer to it as Schiff's reagent or fuchsin-sulphurous acid.

It seems to make little difference as to the source of the sulphurous acid. As this is simply sulphur dioxide dissolved in water there are several ways to obtain it. The four procedures that have been recommended are:–

+ H2SO3 =
Note the loss of the quinoid ring of pararosanilin in the left diagram (highlighted in red) by the change from double to single bonds because of the addition of -SO3H and the formation of Schiff's reagent.

 

Schiff's reagent should be colourless or very pale yellow. However, the pararosanilin can contain other dyes, particularly if it is from a sample of basic fuchsin, which is a mixture. The other dyes present are usually homologues of pararosanilin, such as rosanilin and magenta II, but produce deep amber solutions and may color aldehydes brownish red. These darker products can be removed by adding a small amount of activated charcoal powder, shaking the solution for about a minute and filtering. Sometimes this treatment doesn't work and the solution remains brown. Unfortunately, if enough charcoal is then added to completely remove all the brown discolouration, the resulting clear solution may only give very pale staining. Usually this means that a sample of basic fuchsin was used which contains large amounts of one of the other dyes. If possible buy samples of basic fuchsin specified as suitable for Schiff's reagent, or buy pararosanilin.

As previously mentioned, Schiff's reagent combines with aldehydes to give a bright red product. Histologically, the aldehydes are either attached to, or produced from, a tissue structure. Therefore the tissue structure itself becomes coloured bright red. The mechanism is the same for all aldehydes in tissues. The aldehyde condenses with the Schiff's reagent to make a new compound attached to the tissue. In the process the chromophore reforms, and colour is produced.

+ 2(R-CHO) =
The single bonds of Schiff's reagent shown in the leftmost diagram (highlighted in red), reform into a quinoid ring in the product shown in the middle diagram. This is due to the addition of aldehyde. The formula on the right is a possible alternative product.

 

The new red product is shown with two formulas. Most explanations give the first as the final product. However, some texts present an alternative view incorporating one aldehyde. In either case the dye is composed of the Schiff's reagent, the aldehyde and the chemical to which the aldehyde is attached. Kiernan reports that the latest investigations indicate the product is different from that given above, and exists as a tautomer of the two products below.

 

Other dyes
Dyes other than pararosanilin can be used to make Schiff-type reagents. These are often referred to as pseudo-Schiff reagents. Usually these dyes do not decolorise completely, although the colour and translucency of the solution is often altered. It is possible that residual coloured compounds in these solutions can stain tissues ionically. In order to remove this staining, it is customary to treat sections with acid alcohol afterwards. Any staining left after acid alcohol treatment is a positive reaction from the pseudo-Schiff reagent.

None of these pseudo-Schiff's reagents have gained anything approaching the popularity of pararosanilin Schiff's reagent, and this solution remains the standard. The reason is the bright and clear red staining by pararosanilin Shiff's reagent. The other dyes give inferior colouration, although a few are reasonably effective. The solutions that have gained the most favour are those made with fluorescent dyes, acriflavine (see below), for example. These enable a fluorescent positive result which can be very useful for demonstrating materials such as fungi, for instance, which may be present in small amounts.

Culling gives a list of many of these dyes, a few of which are listed here. For more information refer to Kasten's papers below, quoted by Culling.

Dye CI Number Colour   Dye CI Number Colour
Acid fuchsin 42685 violet Acriflavine 46000 yellow
Azure A 52005 blue Azure C 52005 blue
Crystal violet 42555 blue-violet Methyl violet 42535 violet
Methylene blue 52015 blue Safranin O 50240 red
Thionin 52005 blue Toluidine blue 52040 blue

 

Sulphite rinses
When tissues are removed from Schiff's reagent and washed, there is inevitably some carry over to the washing fluid. If tap water is used there is a rapid recolouring of the reagent and the water turns quite red. Initially, there were some concerns that this recoloured Schiff's reagent would behave as a basic dye and stain the tissues, giving false positives. For that reason sulphite rinses were recommended. These are dilute sulphurous acid, and are used to wash off the Schiff's reagent for a few minutes, diluting it out well enough before washing the tissues in water, that there is no likelihood of non-aldehyde staining. Experience over many years has shown that these rinses are not necessary and washing well with tap water is satisfactory, provided the sections do not stay in recoloured Schiff's reagent.

Sulphite rinses (Prepare at the time of use)
Water 90 mL
Potassium metabisulphite 10% aqueous 5 mL
N1 hydrochloric acid 5 mL

 

If a pseudo-Schiff's reagent is used, then the sections should be treated with 1% hydrochloric acid in 70% ethanol for 5-10 minutes after rinsing it off. Some of these dyes may not be completely decolorised and may stain ionically. The acid alcohol will remove any such staining. After the acid alcohol treatment the sections should be washed with tap water as usual.

 

Aldehydes
Although Schiff's reagent is used to detect aldehydes, these are not usually found free in tissues and must be produced in some fashion. There are three ways this can be done. The first, and by far the commonest, is to oxidise certain of the tissue components. This produces aldehyde groups, enabling the material to which the component is attached to be demonstrated. The second way is to treat sections with acids to convert some of the deoxyribose in DNA to aldehydes and then colour them with Schiff's reagent. The third is to attach an aldehyde directly to the tissues, usually a protein, then demonstrate the aldehyde with the Schiff's reagent.

This latter can be seen easily with glutaraldehyde fixation for light microscopy. Glutaraldehyde has two aldehyde groups and, if the molecule attaches to the tissue by one of them, the other remains unattached when fixation is complete. Due to this, with any procedure using schiff's reagent the unattached aldehydes will react with it to produce a deep pink background. So, before Schiff's reagent can be applied to these sections, the aldehydes should be blocked so they cannot react.

In any critical application using this reagent, two controls must be used. The first is a section to which an aldehyde block has been applied. The second is a section in which the Schiff's reagent has been applied to the tissue without any pretreatment. The first section thus blocks any pre-existing aldehydes, while the second identifies where they were located. In addition, of course, known negative and positive control sections for the target tissue element should be employed. If these two latter controls do not stain as expected, then the Schiff's reagents, and any others, should be examined to ensure they have not deteriorated, and are suitable for use.

 

References
Pearse, A. G. E., (1968, 1972)
Histochemistry: Theoretical and Applied, Ed. 3
Churchill Livingstone, Edinburgh, London, UK

Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.

Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
  Citing:–
    Kasten, F.H., (1958), Stain Technology, vol.33, pp.39
    Kasten, F.H., (1959), Histochemie, vol.1, pp.466

Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.

 


 

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