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Fixation

Fixatives and Preservatives are used to preserve tissues for their study. Common fixatives such as neutral buffered formalin (10% formaldehyde in Phosphate buffered saline (PBS)) are also used to study the structures of the cell, and the cell organelles found in the individual cells (e.g., nucleus, rough endoplasmic reticulum,and mitochondria). The tissues must be first mechanically and biochemically stabilized in a fixative. It is important to consider that a fixative should not be too toxic to its handler, and it should not damage the tissue being preserved.

Fixation Table of Contents

Introduction to Tissue Fixation and Histology

When a tissue is removed from a living condition, several changes are initiated in its cells. Bacteria begin to mtiltiply and destroy them. Also autolysis (self-digestion), a lysis or dissolution of the cells by contained enzymes, sets in. The enzymes appear to reverse their action; instead of synthesizing amino acids into cell proteins, they begin to split proteins into amino acids. These amino acids diffuse out of the cells, and as a re sult proteins no longer are coagulable by chemical reagents. The above changes lead to so-called post-mortem conditions. For purposes of lab oratory examination it is necessary to treat the tissue to prevent these post-mortem effects; it is also necessary to convert the cell parts into materials that will remain insoluble during subsequent treatment, and to protect the cells from distortion and shrinkage when subjected to fluids such as alcohols and hot paraffin. Further important objectives of tissue preparation are to improve the staining potential of tissue parts and to change their refractive indices toward improved visibility. The procedure used to meet the above requirements is called fixation and the fluids are called fixatives or fixing solutions. Fixing solutions should meet the following principal objectives:

Fixation Purpose:

The purpose of fixation is to:

  • 1. Penetrate rapidly to prevent post-mortem changes.
  • 2. Coagulate cell contents into insoluble substances.
  • 3. Protect tissue against shrinkage and distortion during dehydration, embedding and sectioning.
  • 4. Allow cell parts to become selectively and clearly visible by means of dyes and improved refractive indices.

Fixatives

In some cases the fixative may have a mordanting (combining insolubly) effect on the tissue, thereby bringing the two together in a staining action and assisting in the attachment of dyes and proteins to each other.

Tissues must be placed in fixatives as soon as possible after death. If, however, delay is unavoidable, they should be placed in a refrigerator, thus reducing autolysis and putrefaction to a minimum until the fixa tive can be applied. A single chemical seldom possesses all of the requisite qualities of a good fixative; a fixing solution therefore is rarely composed of only one chemical. A familiar exception is formalin. Other reliable fixatives con tain one or more chemicals to coagulate the proteins of the cells, and one or more chemicals to render the proteins insoluble without actu ally coagulating them.

Coagulant fixatives change the spongework of proteins into meshes through which paraffin can easily pass, thus form ing a tissue of proper consistency for sectioning. In addition, the pro tein linkages are strengthened by coagulants against breaking down during later procedures. Used alone, however, coagulants have disadvan tages in that they may form too coarse a network for the best cytological detail. Also, coagulation tends to induce the formation of artificial structures (artifacts). Noncoagulant fixatives produce fewer artifacts but if used alone have the disadvantage of giving the tissue a poor con sistency for embedding.

It therefore follows that the ideal fixing fluid is a combination of one or more protein coagulants and one or more non coagulants. The most efficient solutions are of this nature, and combine if possible all types of action. This excludes those designed for the fixation of some specific cell component, such as chromosomes, glycogen, mitochondria.

Fixation Protocols

Because they contain ingredients which act upon each other, many mixtures are most efficient when made up fresh.

However, the individual parts usually can be made up as stock solutions and mixed together immediately before use. Among the frequently used chemicals are form aldehyde, ethyl alcohol, acetic acid, picric acid, potassium dichromate, mercuric chloride, chromic acid and osmium tetroxide.

Since every chemical possesses its own set of advantages and disadvantages, each solution component should, whenever possible, compensate for a defect in some other component. For example, in the case of the widely used fixative, Bouin's solution: 1. Formaldehyde fixes the cytoplasm but in such a manner that it retards paraffin penetration. It fixes chromatin poorly and makes cytoplasm basiphilic. 2. Picric acid coagulates cyto plasm so that it admits paraffin, leaves the tissue soft, fixes chromatin and makes the cytoplasm acidophilic, but it shrinks and makes chroma Chemicals Commonly Used in Fixing Solutions 5 tin acidophilic. 3. Acetic acid compensates for both the latter defects. No hard and fast rule exists concerning the choice of a fixative; gen' erally selection is determined by the type of investigation.

If there is any question as to future plans for a tissue, formalin is a safe choice; it permits secondary or post-fixation. Some suggestions can be included. If over-all anatomy of the tisstie is satisfactory, the rotitine fixatives can be used: formalin, Siisa, Zenker, Helly, or Bouin. Special fixatives for cell inckisions are Carnoy, Flemming, Champy, Helly, Schaudinn, Regand, and others. For histochemistry the researcher is limited to formalin, acetone, or ethyl alcohol. Most fixing solutions are named after the person originating them; Zenker and Bouin are examples. If the same man originated more than one combination of chemicals, additional means of designating them have been used. Flemming's weak and strong solutions, Allen's B3, Ely series, etc. Susa fluid was named by Heidenhain after the first two letters of two words: sublimate and saute.

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