Iron Resorcin Dye Methods
The iron-resorcin-fuchsin variants, of which there are several, are quite popular for the demonstration of elastic fibres. Usually, the results are well stained elastic fibres of all thicknesses in a single section. Weigert's original technique used basic fuchsin, but several other dyes, alone and in combination, have also been shown to give effective staining in different colours which make the fibres more prominent. Often, depending on the method, a Mallory bleach may be required before the staining solution is applied.
Black, or near black staining of elastic fibres is often considered desirable, either because it is reminiscent of Verhöeff's iron hematoxylin technique, which is so popular, or because it contrasts so well with many counterstains. Miller's variant produces black stained fibres with overnight staining and Humberstone's produces blue-black fibres, and may be somewhat faster with an older solution.
Most of these methods use resorcinol but at least one uses orcinol. Orcinol is the compound used as the starting point for making synthetic orcien. It is also worth noting the relationship these compounds have with phenol, as at least one method incorporates it into the staining solution.
The compounding procedure for iron-resorcin-dye solutions is to combine the dye with the resorcinol, bring to the boil, then add ferric chloride. A precipitate of unspecified composition forms which is presumed to be a complex of the resorcinol and dye, possibly with iron present. The precipitate is collected by filtration, dried, then redissolved in ethanol, often with some acid added. Depending on the dye or dye combinations used, this solution is stable from a few months to several years.
Some variants replace part of the dye content, or add to it, by incorporating dextrin into the solution. Dextrins are partially hydrolysed starch with varying numbers of glucose units. They are probably incorporated into the compound which is precipitated along with the resorcinol and dyes, but the composition is unspecified again. It is presumed that the dye, the resorcinol and dextrin, if added, combine to form large molecules which are precipitated by the ferric chloride, possibly with some chemical attachment by the iron. The large molecules formed are prone to van der Waal's forces and attach to the elastic fibres by them.
Ferric chloride is available in two solid forms, as an anhydrous powder and as hydrated crystals (FeCl3.6H20). Usually, the ferric chloride used to make the iron-resorcin-dye precipitates is in the form of a 30% solution of hydrated crystals. Occasionally, however, some other concentration is specified, so the directions should be read carefully (compare the orcinol-new fuchsin variant to Humberstone's).
These methods have often been considered tedious to use, since the preparation takes some time. It involves the formation of the dyeing complex, its precipitation, collecting and drying, redissolving it in ethanol with heat, then adding acid. This often takes two days, and the early solutions had a life of only a few months, so the procedure had to be repeated fairly often. Some of the more modern variants are much less tedious to use as they are stable for several years and can therefore be made in larger quantities with the expectation that they will be sufficient for some year's use. I particularly recommend Humberstone's variant. I have used this when it is in excess of ten years old with perfectly satisfactory results. Although the authors say that buffered formalin fixation is not recommended, the present system of short fixation does not appear to inhibit the staining at all.
|Orcien Staining Methods|
Last updated January 2019