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Preparation of Histological Slides
For medical students2 min readUpdated 2026-10-10
Histological slide preparation is a multi-step process of processing biological tissues for microscopic examination. It includes fixation, embedding, sectioning, staining, and mounting, which preserve cellular and tissue architecture.
Section thickness5 µm or more (depending on the microtome)
FixativesFormalin, methanol, and other solutions
MicrotomeInstrument for obtaining ultra-thin tissue sections
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 format in practice.
Smears. Used to study fluid media (e.g., blood or bone marrow).
Imprints. Obtained by pressing an organ (such as the spleen) against a glass slide.
Whole mounts (flat preparations). Intact fragments of thin structures, such as the mesentery or pia mater.
Standard Sectioning Protocol
Creating a histological section is a strictly regulated process consisting of five consecutive steps:
Sampling and fixation. A tissue fragment (approximately $0.5 \times 1 \times 1$ cm) is excised and immersed in a fixative solution (formalin, methanol). Fixation lasts 24 hours and prevents autolysis (self-digestion) through protein denaturation. Afterward, the sample is washed with water.
Dehydration and embedding. To make the tissue hard enough for sectioning, it is impregnated with paraffin or resins. Because paraffin is hydrophobic, the tissue is first dehydrated in ascending concentrations of alcohol (from 70% to 100%), followed by clearing in xylene. The material is then embedded in liquid paraffin (52–56 °C), which solidifies into a 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 to 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 concentrations of alcohol. After staining, the slide is rinsed with water.
Mounting. For long-term storage, the section is dehydrated and cleared again (using carbol-xylene and xylene). A mounting medium (most commonly Canada balsam), whose refractive index is close to that of glass, is applied, and a coverslip is placed on top.
Specialized Methods
In addition to classical paraffin sections, other approaches exist:
Rapid intraoperative diagnostics (frozen sections). Used during surgery or for histochemistry. The sample is frozen (using liquid nitrogen or carbon dioxide) and sectioned in a cryostat. This bypasses fixation, alcohol dehydration, and embedding, providing immediate results.
Whole mounts and smears. These preparations omit the embedding and sectioning steps. The cycle includes only fixation, staining, and mounting. Smears are often mounted using cedar oil instead of Canada balsam.
Frequently asked questions
What fixing fluids are used for preparing histological specimens?
Simple and complex fixatives are used to prevent tissue decomposition and autolysis. These include:
Simple fixatives — formalin, alcohol (methanol), acids, and heavy metal salts. Osmium tetroxide is used for 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?
The combination of hematoxylin and eosin is the standard routine stain.
Hematoxylin — a basic dye; basophilic structures stain with basic dyes. These include cell nuclei, ribosomes, and the amorphous ground substance of the extracellular matrix.
Eosin — an acidic bright pink dye; acidophilic structures stain with acidic dyes. These include cytoplasmic proteins (such as erythrocyte hemoglobin) and non-cellular structures like collagen fibers.
Other stains include acid fuchsin, azure II, carmine, and neutral stains (mixtures of basic and acidic dyes).
What media are used for mounting histological slides?
Mounting media are used to preserve slides; their refractive index must be close to that of glass. These include:
Canada balsam — a classic mounting medium used under a coverslip.
Glycerol jelly and polystyrene — mounting media for various sections.
Cedar oil — frequently used for smears instead of Canada balsam.
Why is tissue fixation necessary?
Fixation is necessary to prevent autolysis (tissue self-digestion) by cellular enzymes. Fixatives cause protein denaturation, preserving the native structural state.
Why must tissue be dehydrated before paraffin embedding?
Paraffin is hydrophobic and cannot penetrate water-containing tissue. Therefore, water is first replaced by alcohol and then by an intermediate clearing solvent (xylene).
What is the purpose of rapid intraoperative diagnostics?
This is a method where tissue is rapidly frozen and sectioned in a cryostat. It eliminates lengthy fixation and embedding steps, allowing for a fast diagnosis directly during surgery.
Go deeper
Mechanisms of chemical protein denaturation during fixation
Properties of various embedding media (celloidin, synthetic resins)
Design and operating principles of the microtome
Types of dyes and mechanisms of their tissue interaction