Histology. Research methods in histology. Preparation of histological specimen

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Histology. Research methods in histology. Preparation of histological specimen

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Topic 2. RESEARCH METHODS IN HISTOLOGY. PREPARATION OF HISTOLOGICAL PREPARATION

The main research method in histology is microscopy - the study of histological preparations under a microscope. Recently, microscopy has been combined with other methods - histochemistry and historadiography. For microscopy, various designs of microscopes are used, which allow studying various parameters of histological preparations.

The following types of microscopy are distinguished:

1) light microscopy (the most common type of microscopy, while the resolution of the microscope is 0,2 microns);

2) ultraviolet microscopy (resolution of the microscope is 0,1 microns);

3) luminescent microscopy (used to determine certain chemical structures in the histological specimen under study);

4) phase contrast microscopy (used to detect and study certain structures in unstained histological preparations);

5) polarizing microscopy (used mainly to study fibrous structures);

6) dark field microscopy is used to study living objects;

7) incident light microscopy (designed to study thick objects);

8) electron microscopy (the most modern type of microscopy with a resolution of 0,1 - 0,7 nm). There are two types of electron microscopy - transmission (transmission) and scanning (or solution) microscopy, which displays surface ultrastructures.

Histological and cytochemical methods are used to determine the composition of chemicals and their amount in certain structures. The principle of the method lies in the chemical reaction between the reagent and the substrate contained in the test substance. In this case, the resulting reaction by-products can be detected using light or luminescent microscopy.

The method of histoautoradiography makes it possible to reveal the composition of chemicals in the structures under study and the intensity of the exchange by the inclusion of radioactive isotopes. This method is most often used in animal experiments.

The interferonometry method makes it possible to determine the dry mass of a substance in living or fixed objects.

The cell culture method is the cultivation of cells in test tubes or in special capsules in the body and the subsequent examination of living cells under a microscope.

The method of vital staining is the introduction of a dye (trepan blue) into the blood or into the abdominal cavity of the animal, which during the life of the animal is captured by certain cells - macrophages, and after the slaughter of the animal and the preparation of the drug, cells containing the dye are determined and counted.

Immunomorphological methods allow using preliminary immune reactions (based on antigen-antibody interaction) to determine the subpopulation of lymphocytes, the degree of foreignness of cells, to carry out histological typing of tissues and organs, i.e., to determine their histocompatibility for further transplantation.

The differential centrifugation method is the study of individual organelles or even their fragments isolated from a cell. To do this, a piece of the organ under study is rubbed, filled with saline, and then dispersed in a centrifuge at various speeds (from 2 to 150 thousand per 1 min). As a result of centrifugation, fractions of interest are obtained, which are then studied by various methods.

Methods of morphometry - quantitative methods. They allow you to determine the size and volume of the nucleus - karyometry, cells - cytometry, organelles - electronic morphometry, as well as determine the number of cells of various populations and subpopulations. These methods are widely used in scientific research.

Various experimental methods - food and water load, physical methods (UHF, microwave, lasers, magnets). They are used to study the reaction of structures of interest to a particular impact and are combined with the methods of morphometry, cyto- and histochemistry. These methods are also used in scientific research.

Thus, the main and most common method of study in histology is microscopy. Preparation of a histological preparation includes the following steps.

1. Taking material - a piece of tissue or organ. When taking material, the following rules must be observed:

1) sampling should be carried out as soon as possible after the death or slaughter of the animal, if possible from a living object, in order to preserve the structure of the studied cells as best as possible;

2) the sampling of the material should be carried out with a sharp instrument so as not to injure the tissues;

3) the thickness of the piece should not exceed 5 mm so that the fixing solution can penetrate the entire depth of the tissue;

4) it is necessary to mark the piece, indicating the name of the body, the number of the animal or the name of the person, the date of sampling.

2. Fixing the material. This stage is carried out in order to stop the metabolic processes in the cell and save it from decay. To do this, a piece of tissue taken for examination is immersed in a fixing solution. The solution can be simple (alcohol or formalin) and complex (Carnoy's solution, Zinker's fixative). The fixative causes protein denaturation and keeps the cell structure in a state close to life. Fixation can also be carried out by freezing - cooling with liquid nitrogen or a jet of carbon dioxide.

3. Pouring tissue pieces into sealing media (paraffin, resins) - or freezing. This stage is necessary in order to subsequently make a thin section of the tissue under study.

4. Preparation of sections on a microtome or ultramicrotome using special knives. After that, sections for light microscopy are glued to glass slides, and for electron microscopy, they are mounted on special grids.

5. Staining of sections or their contrasting (for electron microscopy). Before staining the sections, it is necessary to remove the sealing medium - to perform deparaffing. With the help of coloring, the contrast of the studied structures is achieved. Dyes can be divided into basic, acidic and neutral. The most widely used basic dyes (hematoxylin) and acidic (eosin). Complex dyes are also often used.

6. Section clearing in xylene and toluene. They are encapsulated in resins (balm and polystyrene) and covered with a coverslip.

After these procedures, the drug can be examined under a light microscope. Light microscope sections placed under glass can be stored for a long time and reused. For electron microscopy, each section is used only 1 time, while it is photographed, and the study of tissue structures is carried out according to the electron diffraction pattern.

If the tissue has a liquid consistency (for example, blood, bone marrow), then the preparation is made in the form of a smear on a glass slide, which is then also fixed, stained and studied.

From brittle parenchymal organs, preparations are made in the form of an organ imprint, this organ is fractured, then a glass slide is applied to the fracture site, on which free cells are glued. After that, the drug is fixed and studied.

From some organs (for example, the mesentery, pia mater) or from loose fibrous connective tissue, film preparations are made by stretching or crushing between two glasses, followed by fixation and pouring into resins.

Authors: Selezneva T.D., Mishin A.S., Barsukov V.Yu.

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