Appendix 1: General Principles of Histotechnology
The simplest method for the direct observation of living cells is to remove them from the organism, place them on a slide and examine them by dark-field or phase-contrast microscopy or light microscopy after staining.
However, living tissues and cells are difficult to examine microscopically; instead, they are usually killed by careful fixation to minimize alterations of in vivo morphology. Subsequent processing ends with tissues embedded in a material which facilitates thin sectioning; for light microscopy this is usually paraffin. The steps for processing tissue for histology are as follows.
- Removal of the tissue.
- Fixation. The sample, preferably small, is placed in a reagent (e.g. formalin which is 10% aqueous solution of 40% formaldehyde) which preserves substances and structures in the cells and tissues and prevents autolytic changes. Depending on the size of the piece, it should be fixed from 4-24 hours.
- Washing and Dehydration. After fixation the specimen is washed in water to remove the excess fixative then placed in increasing strengths of alcohol or other dehydrating agents. (So the original water in the tissue and what was in the water and not precipitated by the formalin can be removed by the dehydrating agent – eg. edema fluid with low level albumin/protein).
- Clearing. This is the process of removing the dehydrating agent with some fluid which is miscible both with the dehydrating agents and with the embedding medium. (Since a common embedding agent is paraffin which is an oil, clearing agents often replace oils that are not precipitated by formalin – eg. triglycerides in fat cells).
- Infiltration and Embedding. In this process the clearing agent is replaced with the embedding medium, usually paraffin.
- Cutting. Slicing and removing of 3 and 10 micrometers (microns) – thick sections of the tissues with a microtome.
- Section Mounting. It is accomplished by transferring the sections to a clean glass microscope slide.
- Deceration. This is the removal of the embedding agent.
- Staining. This is a process of increasing the visibility of cells by the application of dyes or by their reaction with chemical agents to form visible substances.
- Mounting. This is the act of covering the cleared section with a drop of mounting medium and a thin glass cover-slip. Mounting media have the same indices of refraction as glass and harden as the solvent evaporates, thus making the preparation permanent.