Types of histological specimens
Depending on the preparation method, there are four main types of specimens:
- Sections. Thin slices of organs. This is the most common option in clinical practice.
- Smears. Used to study fluid media (e.g., blood or bone marrow).
- Imprints (squashes). Obtained by pressing an organ (e.g., the spleen) against a glass slide.
- Whole mounts. These are intact fragments of thin structures, such as the peritoneum or pia mater.
Standard sectioning workflow
Creating a histological section is a strictly regulated process consisting of five consecutive steps:
- Sampling and fixation. A fragment of the organ (about $0.5 \times 1 \times 1$ cm) is excised and immersed in a fixing solution (formalin, methanol). Fixation lasts for 24 hours and prevents autolysis (self-digestion) through protein denaturation. Afterward, the sample is washed with water.
- Dehydration and embedding. To make the tissue firm enough for sectioning, it is impregnated with paraffin or resins. Since paraffin is hydrophobic, the tissue is first dehydrated in graded alcohols (from 70% to 100%), after which the alcohol is displaced by xylene. The material is then embedded in liquid paraffin (52–56 °C), which solidifies into a firm block upon cooling.
- Microtomy (sectioning). The block is mounted in a microtome. The instrument cuts thin layers (e.g., 7 µm), which are then flattened in warm water and transferred onto a glass slide.
- Staining. Before applying a water-soluble dye, the tissue must be deparaffinized: paraffin is removed with xylene, and water is reintroduced through descending grades of alcohol. After staining, the specimen is washed with water.
- Mounting. For long-term storage, the section is dehydrated again and cleared (using carbol-xylene and xylene). A mounting medium (most commonly Canada balsam) with a refractive index close to glass is then applied, and the specimen is covered with a coverslip.
Features of other methods
In addition to classic paraffin sections, other approaches exist:
- Urgent diagnostics (frozen section). Used during surgery or for histochemistry. The sample is frozen (using liquid nitrogen or carbon dioxide) and sectioned in a cryostat. This eliminates the need for fixation, alcohol dehydration, and embedding, providing immediate results.
- Whole mounts and smears. Their preparation bypasses the embedding and sectioning steps. The cycle includes only fixation, staining, and mounting. Furthermore, smears are often mounted in cedar oil rather than Canada balsam.
Briefly: answers to topical questions
What fixatives are used to prepare histological specimens?
Simple and complex fixatives are used to fix histological specimens, preventing tissue decomposition and autolysis. These solutions include:
- Simple fixatives — formalin, alcohol (methanol), acids, and heavy metal salts. Osmic acid is used for the specific fixation of lipids and myelin nerve fibers.
- Complex fixatives — various mixtures of simple fixing solutions.
Which stains are most commonly used for routine histological staining?
Sources indicate that the combination of hematoxylin and eosin is the standard staining method for histological specimens.
- Hematoxylin — a basic dye; basophilic structures are stained with basic dyes. These include cell nuclei and ribosomes, as well as the amorphous component of the extracellular matrix.
- Eosin — an acidic bright pink dye; oxyphilic structures are stained with acidic dyes. These include cytoplasmic proteins, including erythrocyte hemoglobin, and non-cellular structures such as collagen fibers.
Sources also list other stains: acid fuchsin, azure II, carmine, and neutral dyes; neutral dyes are a mixture of basic and acidic dyes.
What mounting media are used for histological specimens?
Preservative media are used to mount histological specimens; the refractive index of such a medium must be close to that of glass. Sources indicate:
- Canada balsam — an example of a mounting medium for coverslipping a section.
- Glycerin gelatin and polystyrene — mounting media for sections.
- Cedar oil — often used instead of Canada balsam for smears.
Class and exam questions
What is the purpose of tissue fixation?
Fixation is necessary to prevent autolysis (self-digestion) of tissues by cellular enzymes. Fixatives cause protein denaturation while preserving the vital structure.
Why must tissue be dehydrated before paraffin embedding?
Paraffin has hydrophobic properties and cannot penetrate water-containing tissue. Therefore, water is first replaced by alcohol and then by an intermediate solvent (xylene).
What is the essence of urgent diagnostics?
This is a method in which tissue is rapidly frozen and sectioned in a cryostat. It eliminates lengthy fixation and embedding steps, allowing for rapid specimen preparation directly during surgery.
What else to study on this topic
- Mechanisms of chemical protein denaturation during fixation
- Properties of various embedding media (celloidin, synthetic resins)
- Design and operating principle of the microtome
- Types of dyes and mechanisms of their interaction with tissues
- Detailed algorithm of histochemical studies
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