Trapping Agents

Trapping agents are chemicals which inhibit removal of dyes from tissues. Rather than stop removal of the dye completely, the usual purpose is to slow it down so that tissues which are more intensely stained still retain large amounts of dye when the background has been decolourised. In this way contrast can be enhanced.

They are often confused with mordants, although their function is completely different.

Trapping agent   Mordant
Usually non-metals. Metals with a minimum valency of two.
No single reaction type. Form co-ordination compounds with dyes.
Usually used with basic dyes. Used with specific acid dyes.
Usually applied after the dye. Usually applied before or with the dye.
Not required to complete staining. No staining possible if not used.
Inhibits removal of dye. may be used to remove dye.

 

By far the commonest example of a trapping agent is iodine in a Gram stain. Picric acid has also been used for this purpose in the Gram-Weigert method, and sodium chloride has been used in a Salt Gram technique. There are also methods for other structures that use tannin or tannic acid as trapping agents, but they are not very common.

With iodine, salt and picric acid the basic principle is that the trapping agent forms an insoluble compound with the dye (crystal violet) which precipitates within a structure, perhaps in combination with some tissue constituent. A solvent is applied in which the precipitated dye is soluble. The dye dissolves out easily from most areas, but its dissolution is resisted for some reason in other structures. The solvent is removed before these resistant areas are affected. They then appear deep violet on a relatively colourless background.

These techniques are not used solely for bacteria. They are also suitable for the demonstration of fibrin, keratin, and other structures if the decolouriser is chosen with care. For bacteria, acetone is usually used because it removes the dye very rapidly and thoroughly. For non-bacterial demonstrations, aniline-xylene is often used as it is a gentler solvent which extracts dye more slowly than acetone and its like. This gives more control over the process and enables other structures to be demonstrated.

The reasons for retention of the dye may vary. There has been some disagreement as to the basis for Gram staining of bacteria. With other structures, such as fibrin or keratin, however, it seems to be just that more dye is present in them than in other tissues. It therefore takes longer to remove it all. We can just stop extracting dye when the staining is satisfactory. A trapping agent in conjunction with a slow solvent simply accentuates this disparity.