The first and most critical step in the 10 Histotechnological processes. It involves fixing and preserving fresh tissue for examination.
Fixation
Killing is necessary to stop the metabolic processes that continue to alter the state of tissue to be examined
There is not one fixative that solves all fixation processes because there are multiple fixing agents available in the market
The container is actually three-fourths full which is the ideal volume for us to meet the fixation process
No process of histotechnology is more critical to slide preparation than fixation. If you got fixation wrong, then you would expect that the succeeding processes will not be adequate.
Primary aim of fixation
Cells and its surrounding tissues should be preserved in the morphologic and chemical integrity of the cell in as life-like manner as possible
Secondary aim of fixation
To harden and protect the tissue from the trauma of further handling
Degeneration
The state or process of being or becoming degenerate; decline or deterioration. Deterioration and loss of function in the cells of a tissue or organ.
Decomposition
The process by which organic substances are broken down into a much simpler form of matter
Putrefaction
Decomposition of proteins in a process that results in the eventual breakdown of cohesion between tissues and the liquefaction of most organs. It is one of the seven stages in the decomposition of the body after death.
Distortion
The alteration of the original shape (or other characteristic) of something
Additive fixation
The chemical constituent of the fixative is taken in and becomes part of the tissue
Non-additive fixation
The fixing agent is NOT taken in, but changes the tissue composition and stabilizes the tissue by removing the bound water attached by the hydrogen bonds
Coagulant fixatives
Acts by creating a network that allows solutions to readily penetrate the interior of the tissue
Non-coagulant fixatives
Creates a gel that makes it difficult to penetrate by subsequent solutions. Tissues subjected to non-coagulant fixatives must be cut thinly.
Factors involved in fixation
Hydrogen ion concentration (satisfactory between pH 6.0 and 8.0)
Temperature (commonly room temperature between 20-24°C, electron microscopy 0-4°C)
Thickness of section (electron microscopy 1 to 2 mm^2, light microscopy 2 cm^2, no more than 0.4 cm)
Osmolality (best using slightly hypertonic solution 400-450 mOsm)
Formalin should have a duration of 2-6 hours during the day the specimen is obtained. Prolonged fixation to formalin will result in shrinkage and hardening of tissue, and inhibit enzyme activity and immunologic reactions.
Practical considerations of fixation
Speed (tissue must be fixed immediately to prevent autolysis and putrefaction)
Penetration (commonly 1 mm per hour, tougher tissues need more penetration)
Volume (fixative must be 10-25 times greater than tissue volume, ideal ratio 1:20)
Duration (fibrous organs need longer fixation than small/loose tissues, can be reduced by heat, vacuum, agitation, or microwave)
Methods of fixation
Chemical fixation
Vapor fixation
Heat fixation
Microwave irradiation
Ultrasound fixation
Brain cells are very sensitive once they have been harvested and deteriorate very quickly so when we receive a section of the brain or the entire brain, it must immediately be fixed in the fixative lasts for about 2-3 weeks.
Bone marrow continues to undergo mitosis up to 30 minutes after death when refrigerated.
General effects of fixatives
Harden soft and friable tissues
Make cells resistant to damage and distortion
Inhibit bacterial decomposition
Increase optical differentiation of cells and tissue components
Acts as mordants or accentuators or may inhibit certain dyes