BIOLOGY Volume 1 - A Guide to General Biology - 2004

5. CELLS

5.12. Microscopy Techniques

5.12.2. Permanent Mounts

Fixation

Fixation is the preservation of material in a state as close to its natural condition as possible. This requires the rapid killing of Tissues, which is best achieved by using small pieces of living material. The substance used for this purpose is called a fixative. Rapid fixation preserves the initial Structure OF THE specimen while hardening the tissues sufficiently to allow thin sections to be cut.

Dehydration

Dehydration is carried out when preparing material for embedding or for mounting in an appropriate medium (see below) that is not miscible with Water. Water must also be removed because otherwise the specimen would eventually be destroyed by Bacteria. To preserve ultrastructure, dehydration must be carried out gradually by treating the material with a series of aqueous solutions of ethanol or propanone (acetone) of steadily increasing concentration, finishing with "absolute" (anhydrous) ethanol or propanone.

Clearing

Some of the commonly used embedding and mounting media are not miscible with alcohol. Therefore, the alcohol must be gradually replaced by a medium (the clearing agent) with which the embedding medium is miscible, such as xylene. This process also makes the material transparent.

Embedding

To obtain very thin sections using a microtome, the material must be embedded in an appropriate supportive medium. When preparing specimens for light Cell/15.html">Microscopy, objects are embedded in paraffin wax, which is then allowed to cool. For Electron microscopy, harder substances (plastics or resins) must be used, as particularly thin sections are required, which in turn demand a denser supporting matrix.

Sectioning

As a rule, pieces of material are too thick for sufficient light to pass through them for microscopic examination. It is usually necessary to cut a very thin layer of the specimen under study—in other words, to prepare sections. Sections can be cut using a razor blade or a microtome. Manual sectioning is performed using a sharply honed razor blade. For conventional microscopy, sections should be 8—12 μm thick. The tissue is held between two pieces of elderberry pith. The razor blade is moistened with the fluid in which the tissue was stored; the section is cut through both the pith and the tissue by holding the blade horizontally and drawing it toward oneself with a slow, slightly oblique slicing motion. After rapidly making several sections, one should select the thinnest one containing the characteristic tissue regions.

Sections of tissue embedded in a medium can be prepared using a microtome. For light microscopy, sections a few micrometres thick can be cut from paraffin-embedded tissue using a specialized steel knife. Ultramicrotomes are used to produce extremely thin sections (20—100 nm) for electron microscopy. In this case, a diamond or Glass knife is required.

Sections for light microscopy can also be prepared without embedding the material by using a freezing microtome. During the preparation of frozen sections, the specimen is maintained in a frozen and, consequently, solid state.

Staining

As a rule, biological structures in preparations are transparent, so various agents must be employed to provide contrast between them. Staining is the most common method. Some of the stains used in light microscopy are listed in Table 5.5.

Certain stains, at low concentrations, are non-toxic to living tissues and can therefore be used to stain living material. These are known as vital stains. Examples include methylene blue and neutral red.

When staining paraffin sections, the paraffin is removed using a solvent, and the section is partially rehydrated prior to staining.

Class="center">Table 5.5. Stains used for PLANT AND ANIMAL tissues

Mounting

Fully stained sections are mounted on a glass slide in a specialized medium, such as Canada balsam or Euparal; this medium is airtight, allowing the section to be preserved indefinitely. The mounted section is then covered with a coverslip.

The sequence of steps described above is typical when preparing thin sections for permanent mounts. However, the Procedure is frequently modified by the inclusion of the following two steps:

a) if sections of raw material are prepared manually, the section is cut first and fixed afterwards;

b) staining can be performed either after fixation or at some stage during dehydration. For instance, a section can be stained after dehydration in 50% ethanol using a dye dissolved in the same 50% ethanol.

The described slide preparation procedure is largely similar for both light and electron microscopes, although there are some differences in detail (these are listed in Table 5.6).

Table 5.6. Differences in specimen preparation for light and electron microscopes