Any histological or cytological laboratory technique begins with fixation. Fixation is a method of preserving the chemical and physical condition of the cells in the tissue, as well as preventing all biochemical and proteolytic activities inside the cells, such that the cells or tissues can withstand any morphological alteration, distortion, or decomposition after treatment with various reagents. The fixation aids in keeping the tissue in the living system as close to its original state as possible. This is achieved using fixatives. Each fixative has its own set of benefits and drawbacks yet it is difficult finding a widely agreed ideal fixative.
Aims of Fixation
- To preserve the tissue as close to its living state as possible during processing
- To avoid any changes in shape and size of the tissue during processing
- To avoid any autolysis
- To make the tissue firm to hard
- To avoid any bacterial growth in the tissue
- To allow for clear staining
- To improve the optical quality of the cells
Properties of an Ideal Fixative
There are several different types of fixatives on the market. An ideal fixative should be capable of the following:
- It should be capable of preventing autolysis of cells or tissues.
- Should prevent bacterial decomposition of tissue
- Maintain the cell’s volume and shape as much as possible
- Consistently high-quality staining, particularly routine staining such as haematoxylin and eosin staining and Papanicolaou’s staining.
- Immediate action
Changes that occurs in Tissue during Fixation
Fixation can cause the following changes in tissue.
- Changes in Volume: The volume of the cells can be altered by fixatives. Osmium tetroxide, for example, causes cell swelling. The precise mechanism of volume shift is not well known. The volume change may be due to
- Altered membrane permeability
- Inhibition of respiration enzymes
- A change in transport Na+ ions.
Formaldehyde can cause a 33 percent reduction in volume. Glutaraldehyde, on the other hand, causes a lot of tissue shrinkage. When glutaraldehyde and osmium tetroxide are used as fixatives in epoxy resin, however, cell size is increased by 70%.
2. Tissue hardening: Fixation affects the quality of the tissue, and some hardening happens as a result of fixation.
3. Staining interference: Fixation can interfere with enzyme staining. 80 percent of the ribonuclease enzyme is inactivated by formaldehyde. Osmium tetroxide has been shown to prevent the staining of haematoxylin and eosin.
4. Fixation-induced changes in optical density: Fixation can alter the optical density of nuclei, making them appear condensed and hyperchromatic.