Collection of material for microscopic examinations. Fixation

Before a tissue can be looked at, it has to be taken out of the body and stopped from destroying itself. Those are the first two steps of the paraffin technique, the standard route to a permanent histological preparation.

Collection is by biopsy or necropsy. Fixation is the placing of small pieces of tissue in solutions of chemicals that prevent enzyme digestion, which preserves the cell and tissue structure. Fixatives are either simple, a single chemical, or compound, a mixture of simple fixatives.

The first step of the paraffin technique is the removal of tissue, and there are two ways it is obtained:

• biopsy, the removal of tissue from a living patient;
• necropsy, the removal of tissue after death.

The pieces taken are small. That matters for the step that follows: a fixative works by diffusing into the tissue, and a large block would be fixed at its surface and already degrading at its centre.

Fixation is the second step. Small pieces of tissue are placed in solutions of chemicals in order to prevent enzyme digestion. The result is that the cell and tissue structure are preserved.

The problem fixation solves is that a tissue removed from the body is not inert. Its own lysosomal enzymes begin to digest it from the inside, a process called autolysis, and bacteria attack it from the outside. A fixative stops both, and it does so by cross-linking or precipitating the proteins of the tissue so that they can no longer act as enzymes and no longer dissolve away in the solvents used in the later steps.

Everything after this point in the technique, the dehydration, the clearing, the infiltration with hot paraffin, would destroy an unfixed tissue. Fixation is what makes the rest of the procedure survivable.

There are two main groups of fixative: simple and compound.

A simple fixative contains one chemical. Three classes are used:

• aldehydes, for example formaldehyde;
• alcohols, for example methanol;
• oxidizing agents, for example chromic acid.

A compound fixative is a mixed solution of simple fixatives. Two named examples are:

• Carnoy's fixative;
• Flemming's fixative.

Mixtures are used because the simple fixatives have complementary faults. One preserves a structure another one damages, so combining them gives a preparation in which more of the tissue survives recognisably than any single chemical would allow.