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Histological techniques

2. FIXATION



When tissues are obtained from organisms, either living or dead, a degradative process begins. It has to aspects: autolisis (autodigestion) by intracelular enzymes that degrade cells and extracellular matrix, and putrefaction by the degradative activity of microbies (mostly bacteria). Moreover, the methodology of the histological processing to study particular tissular features may degrade or destroy tissular structures. Fixation preserves morphological and molecular features of the tissue as similar as possible as they were in the living organism. It is like taking a picture of the living tissue, keeping it invariable after the histological processing, and observing this picture with the microscope. Thus, fixatives prevent autolisis, protect against microbies degradation, fix soluble molecules, do not cause tissue distortions or retractions, and prepare the tissue for specific staining or study when necessary.

There is no one universal fixative, nor one universal fixation method. Furthermore, several fixatives can be sequentially used if necessary. The selection of fixative and fixation method depends on the features of the tissue that have to be preserved. For instance, if enzymatic activity must be preserved, fixation must not destroy the catalytic center of the enzyme, and then other features of the tissue, like cell morphology, might be modified. The majority of fixatives don not preserve lipids, but these molecules remain in the tissue if solvent solutions are avoided. However, the study of tissue ultrastructure involves organic solvents during the embedding in resins, therefore a fixative that keeps lipids in the tissue is needed if we want to observe cell membranes. Most fixatives do not preserve carbohydrates, but these molecules remain in the tissue because they are usually attached to proteins. Sometimes, some fixatives can modified molecular tissular components so that they are more easily stained by dyes.

In any case, there are some features to be considered before the selection of fixatives:

Diffusion rate. Fixation has to be quick and the diffusion of fixatives through tissues is a limitant feature. The speed of penetration of the fixative determines the size of the sample to be processed. So that, the lower diffusion rate the smaller the sample should be. Furthermore, it also affects the total fixation time: higher diffusion means shorter fixation time.

Fixation rate. This feature does is not a consequence of the fixative diffusion rate, but depends on the chemical nature of the fixative and influences how long the fixation should last.

Hardening. Tissues are usually hardened by fixatives, that depends on the type of fixative and fixation time.

Osmosis and pH. uring fixation, it is important to prevent volume changes in the tissues and cells. These changes may be produced by a different osmolarity or pH between the tissue and the fixative. The alterations of the tissues produced by the histological processing are known as artifacts. Therefore, it is recommended to equilibrate the tissular and fixative osmolarities. It can be accomplished with simple molecules that do no affect the chemical fixation. For instance, 0.9 % NaCl can be used in fixatives for land animals. It is also important to use buffered fixatives with pH similar to that of the tissues.

Mordants. Some tissular structures are difficult to stain because of their low affinity for dyes. This affinity can be increased by treatment before the staining. Some fixatives, besides fixation, can chemically modify the tissues and increases the affinity for some dyes. This type of tissue modification is known as mordant effect.

Artifacts. Fixation may produce tissular alterations like swelling, retraction, crystallization of substances, and movement and extraction of molecules. They may be a consequence of the fixative features or fixation method. The changes introduced in the tissue by the histological process are known as artifacts. It is important to realize that what what we are observing is an artifact by fixation so that it is not described as a tissular feature. Fixatives may produce retractions of the tissue, which can be tested by checking the dimensions of the tissue sample before and after the fixation. However, it should not be assumed that retractions, or swellings, affect all tissues of the sample in the same way.


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Updated: 2018-06-27. 09:02