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Factors Affecting Trichrome Staining

How it Works

There are several factors involved in determining which acid dye attaches to which tissue component. Some of these factors involve the tissue and some the dye, and the more traditional Masson type trichromes also incorporate what are often referred to as polyacids. These factors are all active simultaneously, but in a given circumstance an individual factor may be more or less important. For this reason, the explanations for multi-step and one-step methods differ slightly, although they do depend essentially on the same process.

Learn more about the 3 factors below.

Tissue State

Often, when discussing staining, we focus on the dyes and other chemicals we use in a particular technique. Equally or more important is the reactivity of the tissue that we are staining. There are several factors which may affect the condition of these tissues in relation to the dyes used in staining.


We often do not consider the effect of fixation on staining, but it does have a significant impact. Some fixatives inhibit staining, while others help facilitate it. The choice of fixative can have a significant impact on both the depth of staining achieved and on subsequent manipulations of it.

In the specific context of trichrome stains fixatives containing mercuric chloride and/or picric acid are the ones of choice. Both fixing agents improve staining by acid dyes. Extended fixation in formal sublimate has been recommended for the more difficult methods, used for the demonstration of fibrin.

Reasonably satisfactory staining of material fixed in formalin variants can be obtained with some methods by altering the times of each step, but secondary fixation of sections in Bouin’s fluid or saturated aqueous picric acid for an hour at 56°C is recommended for routine methods whenever possible.

Tissue Accessibility

Obviously, before chemicals, including dyes, can react with tissue they must be able to get there. In some cases the tissue itself inhibits this. There are several reasons why this can be so.

A common cause is the use of an aqueous solvent and a hydrophobic tissue component. It has been suggested that some fibers are coated or intimately associated with lipids of some kind, and these lipid materials inhibit access by aqueous solutions. There are two steps where this can seriously impact on staining. The first is applications of dyes themselves, but perhaps equally important is the inhibition of aqueous fixatives and reduced fixation as a consequence. A process called degreasing has been developed to eliminate both of these problems for methods where fixation and dye application are critical. Other reasons might be the physical state of the tissue, such as dense bone or tendon, which physically inhibits any penetration by dyes. All of these inhibit access of the dye to the tissues, and require increased time to accomplish staining.

Of course, the converse can be true as well. Some tissues can be easily accessed. Collagen is a good example. It is easily stained, and requires little time to accomplish. Between the extremes are a wide variety of “textures”, so to speak, and a correspondingly wide variety of staining results and intensities.

Tissue Density

Tissue density refers to the number of dye binding sites a particular tissue has in a given volume. That is, how much dye will attach to it compared to the amount that will attach to other tissues? Since we are talking about the attachment of acid dyes to tissue, we are really talking about the number of charged amino groups available. Tissues vary in this.

Obviously, if there is more dye attached to a particular structure it will appear more deeply stained. It will also take longer for the dye there to be replaced, and that can be important. Should dye be removed equally from all tissues, those components that have more will retain some dye when other components have been completely decolorized.

Dye State

In solution, dyes do not exist as discrete single molecules. Due to various attractions, such as van der Waal’s forces, they tend to clump together. The larger the molecule, the more likely this is to happen, so that there is some relationship to molecular weight. Unfortunately this is not absolute, and we cannot say that a given dye with a known formula weight has a specific number of molecules in its aggregate.

A second factor is the solvent into which the dye is dissolved. Usually this is water which permits dye aggregates to form quite well. Changing the solvent can affect the aggregate size. When dyes are dissolved in ethanol, the attractions between molecules is less. The propensity to form aggregates in solvents depends to some extent on the polarity of the solvent. Ethanol is a less polar solvent than water, thus the aggregation is lower.

When dyes are applied to tissue the aggregates approach the amino groups. If they are physically able to penetrate the tissue they can react with the amino groups, and the tissue is dyed. It is quite possible that penetration of the dye is inhibited because of its aggregate size, and staining is diminished (usually) or inhibited (rarely) as a consequence.

The difference in dye aggregate size can be used to stain some difficult tissue components. It is commonly used to demonstrate erythrocytes yellow in Lendrum’s Picro-Mallory method for fibrin, for instance. Yellow dyes of low molecular weight are applied in ethanol solution. These easily penetrate and stain erythrocytes. The sections are then washed with water thus changing the solvent. Extraction of the yellow dyes is inhibited because the larger aqueous aggregates have greater difficulty passing the erythrocyte envelope to get out than the smaller aggregates in ethanol did to get in.

Polyacids & Displacement

Displacement is the term used to describe the phenomenon of one charged group being able to replace another similarly charged group in chemical reactions. In practical histological terms, it refers to the ability to replace a dye already attached to tissue groups by another dye which is similarly charged. In other words, within the context of trichrome staining, using one acid dye to replace another on tissue amino groups.

Polyacids are high molecular weight compounds such as tungstophosphoric (phosphotungstic) acid and molybdophosphoric (phosphomolybdic) acid. Most trichrome methods use these in some way or other. There are exceptions, however. The commonest method, van Gieson’s stain, does not use a polyacid at all. The strongly acidic picric acid provides what acidity is required. There is also at least one variation of Lendrum’s Picro-Mallory stain which uses trichloracetic acid rather than one of the acids above. It should be noted that trichloracetic acid differs from the other two in that it does not contain any metal atoms.

Their use is integral to trichrome staining methods, although there is some disagreement as to how they function. The most usual explanation is that they function as if they were very high molecular weight acid dyes. On this basis, they would displace other acid dyes already attached to tissues. Due to the high molecular weight, and the inferred large molecule size, the displacement would first involve the most easily accessible structures such as collagen. While other tissues could be involved, a longer time would be needed for those structures to be affected. This approach envisages that the dye attached to the collagen is removed and replaced by the polyacid. When the next dye is applied it, in turn, replaces the polyacid. This approach results in a sharper contrast between the cytoplasmic staining dye and the collagen staining dye.

The alternate explanation is similar, but the dye applied following the polyacid is considered to link to the tungsten or molybdenum in the tissue rather than to replace it. In this respect it is thought to be similar to, but not the same as, mordant dyeing. This explanation is based on the observation that sections stained with trichrome methods using polyacids can be shown to have tungsten or molybdenum atoms still present in those structures after they have been stained with the subsequent dye.

The first explanation, however, explains why the use of chemicals such as trichloracetic acid can differentiate acid dyes by replacing them. Since this chemical does not have metal atoms it could not participate in a metal-dye complex such as envisioned with the tungsten or molybdenum of the polyacids. It should also be noted that in a mass action approach reactions are not permitted to go to completion, so some residual metal atoms from the polyacids should be expected.

The first explanation is preferred but do not exclude the second.