Atlas of plant and animal histology

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Histological techniques


All tissues obtained from living or dead organisms undergo a degradation process. The degradation may be intrinsic, known as autolysis or autodigestion, which is driven by the activity of lytic tissular enzymes. At the same time, an extrinsic degradation process, called putrefaction, is started by microorganisms, mostly bacteria. Moreover, the methodology of the histological processing to study particular features of the tissue 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. In other words, fixation decreases both molecular and structural changes in the tissues that may happen during the lab processing. It is like taking a picture of the living tissue, keeping it invariable during the histological processing, and observing this picture with the microscope. A correct interpretation of the features of a tissue depends on a proper fixation. For example, a patient may be wrongly diagnosed as a consequence of a bad fixation that produced alterations of the tissular or cellular structures in the biopsy.

Fixation kills cells and should inhibit cell autolisis, protect tissues against microorganisms, insulobilize molecules that can be lost, as well as prevent retractions or swelling of the tissues that may change the tissue morphology. In addition, fixation must prepare the tissue for performing specific histological techniques, if it is necessary, and keep the tissue features unaltered during the lab processing, such as embedding and staining.

Fhere are physic fixatives, such as heat and freezing, and chemical fixatives. Chemical fixatives interact with the tissular molecules. Each type have advantages and disadvantages. Chemical fixatives are the most commonly used in the histology labs for light and electron microscopy studies.

There is no universal fixative, neither only one fixation method. Furthermore, several fixatives can be sequentially used in the same fixation process. The selection of the fixative and fixation method depends on the features of the tissue that need to be preserved. For instance, if enzymatic activity must be preserved, fixation should not destroy the catalytic center of the enzyme, but other features of the tissue, like cell morphology, might be modified. The majority of fixatives do 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 necessary 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 modify molecular components of the tissue so that they are more easily stained by dyes.

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

Diffusion rate. Fixation has to be quick and the diffusion of fixatives through tissues is a limiting factor. How fast the fixative diffuse through the tissues determines the size of the sample to be processed. So that, the lower diffusion rate, the smaller the sample. Furthermore, it also affects the total fixation time: higher diffusion means shorter fixation time.

Fixation rate. This feature 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. Keep in mind that this is a chemical fixation.

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

Osmosis and pH. During fixation, it is important to prevent volume changes in the tissues and cells. These changes may be produced by differences in osmolarity or pH between the tissue and the fixative. Therefore, it is recommended to equal the tissular and fixative osmolarities. It can be accomplished with simple molecules that do not 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.

Temperature. Fixation at higher temperatures than the animal body temperature, or room temperature for plants, decrease the time of fixation. However, it may produce tissue alterations.

Mordants. Some tissular structures are difficult to stain because of their low affinity for dyes. The affinity can be increased by some treatments 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. An artifact is whatever alteration introduced in the tissue during the histological process. Fixation is probably the histological process step with more influence in the final quality of tissues. Unlike staining or a poor paraffin embedding, fixation in irreversible. Depending on the fixative and fixation method, fixation may produce tissular alterations like swelling, retraction, crystallization of substances, and movement and extraction of molecules. These changes may be because of the fixative or the bad use of it. It is important to realize that 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, retractions, or swellings, may not affect all tissues of the sample in the same way.

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