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Mass Action

in Staining

When we discuss the ways dyes and tissues react with one another, we often talk about individual molecules. In reality, of course, a single molecule reacting with another would show us precisely nothing, since it is very unlikely that any system we use could detect it. Histological reactions require millions, perhaps billions, of molecules to take part and to give an observable effect.

A second factor is the variability of tissues. We are not dealing with a homogenous material. As an example, predominantly basic tissues will react with acid dyes, but there may be different degrees of basicness in the one tissue, In other words, some tissues will have more available amino groups than others and can attract different quantities of the acid dye. There are several other factors as well.

Due to these, we have to modify our understanding of the fundamental reactions involved. While it is simple to indicate that amino groups in tissue attach to carboxyl groups on dyes, the carboxyl groups must first reach the amino groups to be able to attach.

Mass action is a way of looking at chemical reactions based on very large numbers of molecules as a whole instead of the reactions between individual molecules. Instead of viewing a reaction between two entities as a single, unchangeable event, it views chemistry as a dynamic process. It is concerned as much with the process of reaching the desired chemical goal, as the goal itself.

Many chemical reactions are reversible. As a consequence, the results of these are often a mixture of the original chemicals and their reaction products in equilibrium. If left to go to completion, the products present depend on the amount of each original reactant.

Mass action is often represented by the equation “A+B ⇔ C+D”. The original compounds are represented by A and B, and their products are represented by C and D. Inherent in this is that the products C and D can interact to produce A and B once again. This goes on continuously, so that all four compounds are present at all times once equilibrium is reached. The amount of each of the four compounds present (i.e. where equilibrium occurs) depends on the amount of A and B that were originally present. To change the equilibrium point, then, is simply a matter of changing the amount of one of the original reactants. This will alter not only its own presence, but the presence of the products derived from it. The equilibrium can also be biased by interrupting the process and removing one of the reactants or products from it. This can occur automatically if one of the products is insoluble, but it is more common in histological staining to simply remove the original reactant.

In histological staining, the original reactants (A and B) are the dye and the complementary tissue groups with which it can react. It might also include any dyes already attached to these tissue groups. The products (C and D) are the complex formed between the colored dye radical and the tissue groups, and a compound from the non-colored component of the dye and displaced atoms or dyes from the tissue group. Histological staining heavily biases this process in favor of one component at a time by applying strong (dye) solutions for a limited time, removing them and then applying other solutions in a series of timed biased events. Equilibrium does not get a chance to develop and is not usually reached, with the possible exception of multi-dye complex solutions (one-step trichromes), although even these are strictly timed.

Equilibrium, in this context, would be total dyeing of the tissue because of the excess dye that is invariably applied. Usually, the process does not go to completion because we limit the time for which the dye is applied, and remove it when we get the degree of staining we want. In many cases, we would consider equilibrium to be gross overstaining. The reversible nature of the process is sometimes difficult to see, but an example would be washing eosin with water after slight overstaining. Here we reduce the amount of one of the original reactants (eosin) and bias the reaction against it, the result being a reduction in the amount of its products (depth of staining).

The point of talking about this subject is to emphasize that the staining process is more than an obligatory reaction between two discrete molecules. It is a dynamic process that can be manipulated in various ways to accomplish surprisingly complex goals.