Cytology, General Histology and Embryology - V. K. Napkhanyuk 2002

Methods of Histological Research
Post-vital Research Methods

The primary object of study using post-mortem (post-vital) Methods is histological slides prepared from fixed structures.

A specimen may appear in the following forms:

— smear (Blood, Bone Marrow, saliva, CEREBROSPINAL FLUID, etc.);

— Touch preparation / impression (Spleen, Thymus, Liver);

— tissue spread (Connective Tissue, Peritoneum, Pleura, pia mater);

— sections (of Organs or Tissues).

Sections are most commonly used in Histology to study The Structure of Cells, tissues, and organs.

Histological specimens (blood smears, touch preparations, spreads, or organ sections) can be examined without special preparation and viewed immediately under a Microscope. However, because biological structures have very low contrast and are poorly visible under a standard Light Microscope, specialized microscopes (phase-contrast, fluorescent, etc.) are used.

Histological studies most frequently utilize specially processed and stained specimens.

The preparation of permanent histological slides requires a very complex process of Sample preparation and consists of several stages:

1. Sampling (tissue harvesting). The sample volume should be within 1 cm³, and the material must be fresh.

2. Fixation of the material is carried out by immersion in a fixing solution. Fixatives can be simple (alcohols, formalin, etc.) or complex (consisting of several simple ones).

3. Washing under running Water for 24 hours to remove the fixative.

4. Dehydration of the specimen in graded alcohol series of increasing concentration (from 60 to 100°).

5. Embedding the specimen in an embedding medium (paraffin, celloidin, synthetic resins).

6. Sectioning using microtomes or ultramicrotomes.

7. Staining or contrasting of sections is performed to make structural details of the specimen distinguishable. Before staining, the embedding medium is removed using xylene, which is subsequently washed out using a descending alcohol series (from 100 to 60°) followed by a water wash. After this, a staining solution is applied to the specimen.

8. Mounting the sections in Canada balsam for long-term preservation of the slides.

To stain specimens, various Dyes are used which, depending on their origin, are classified into plant-based (hematoxylin), animal-based (carmine), and synthetic dyes.

Depending on their chemical properties, dyes are classified as follows: acidic, basic, neutral, and special.

Acidic (anionic) dyes are a group of dyes whose properties are determined by the presence of an acid residue. They stain the Cytoplasm (eosin, light green, etc.).

Basic dyes are cationic dyes, the vast majority of which contain a positively charged nitrogen atom in their structure. They selectively stain Cell nuclei, which is why they are also referred to as nuclear stains (hematoxylin, carmine, safranin).

Neutral dyes are complex compounds formed by aqueous solutions of acidic and basic dyes.

Special dyes are a group of dyes used to identify specific substances, compounds, structures, or tissues. For example, Sudan III (which stains adipose tissue) and orcein (elastic fibers).