Histological research methods are used to study the structure and function of cells and tissues of humans, animals and plants in normal, pathological and experimental conditions. The basis of G. m. and. is - a set of methodological techniques used in the manufacture of preparations of cells and tissues for their microscopic examination. Microscopic study of cells and tissues can be carried out in two main ways, depending on the state of the object under study: the study of living cells and tissues, the study of non-living cells and tissues that retain their structure due to special fixation techniques. The study of living objects - vital (supravital) - makes it possible to observe the physiological processes in cells and tissues, their intravital structure. It is carried out on cells freely suspended in a liquid medium (blood cells, epithelial cells of scrapings, etc.), as well as on cell and tissue cultures grown on special nutrient media. The object of intravital observation can be thin, transparent tissue films (, swimming membrane). In experimental studies, the method of biological windows (implantation of transparent chambers) and the study of tissue implants in a natural transparent environment, for example, in the anterior chamber of the eye of animals, are used. Depending on the task at hand, various special microscopy methods are used in vital studies: dark-field, phase-contrast, fluorescence, polarization, ultraviolet. Vital histological methods are mainly used for biological and biomedical research. Their widespread use is limited by great technical difficulties associated with the properties of surviving tissues. In medical research, especially in the practice of anatomical pathology laboratories, methods of studying fixed objects are used. The purpose of fixation is to preserve the intravital structure of cells and tissues by quickly exposing them to chemical agents that prevent the development of post-mortem changes. The choice of fixation method depends on the objectives of the study and the characteristics of the material to be fixed. Thus, fixing mixtures containing salts of heavy metals (for example, sublimate) are used to identify thin cellular structures. The best fixative for cytological purposes is osmium tetroxide, which is often used in electron microscopy. A universal fixative is formaldehyde, used in the form of a 10% formalin solution. In order to be uniform and complete, the pieces of tissue must be small, and the volume of the fixing liquid must be many times greater than the volume of the material to be fixed. After fixation, the pieces are usually washed in water or alcohol. Solid tissue components (eg, calcium deposits) are removed using decalcification techniques. The fastest and easiest way to prepare a tissue section, which is usually used in express diagnostics, is a piece and obtaining tissue sections on a freezing microtome. However, it is difficult to obtain sufficiently thin sections, as well as sections from small objects and decaying tissues. Therefore, pieces of tissue, as a rule, are poured into sealing media - or celloidin. The fixed is dehydrated in alcohols of increasing strength, passed through an intermediate solvent (xylene or for paraffin, alcohol-ether for celloidin) and impregnated with paraffin or celloidin. in paraffin allows you to get thinner sections (from 5-8 to 1-2 micron) than celloidin. Sections for microscopic examination are prepared using a slide or rotary microtome. Ultra-thin sections (thickness from 90-100 to 5-15 nm) necessary for electron microscopic studies are prepared on an ultratome. Ultratomes are also used in light microscopy to obtain semi-thin sections. The prepared sections are stained to clearly highlight the structures of cells and tissues that perceive dyes differently. Basic dyes - coloring bases and their salts (, toluidine blue, azure, bismarck brown) - stain the so-called basophilic structures (cell nuclei, connective tissue). Acidic dyes - coloring acids and their salts (picric acid, erythrosin) - stain acidophilic, or oxyphilic, structures (cell cytoplasm, collagen, elastic fibers). staining should be distinguished by impregnation - a special method based on certain areas of cells and tissues to restore heavy metals (for example, silver, gold, osmium) from their salts and thereby acquire an intense color. The preparation of a histological preparation is completed by placing it in media that ensure the preservation of the object's structures, its color and transparency. Most often, organic resins are used for these purposes, for example.
1. Small medical encyclopedia. - M.: Medical Encyclopedia. 1991-96 2. First aid. - M.: Great Russian Encyclopedia. 1994 3. Encyclopedic dictionary of medical terms. - M.: Soviet Encyclopedia. - 1982-1984.
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The objects of study can be fixed (dead) or living cells and tissues.
To study the microstructure of raw materials and livestock products, as a rule, fixed cells and tissues are used. Preparations are temporary, intended for a single study, and permanent, which can be stored and repeatedly examined. In addition, total or whole preparations are used.
Temporary drugs can be cooked relatively quickly; for this, the material under study is fixed and sections are obtained on a freezing microtome; in its absence, a thin section of tissue or organ can be made with a scalpel or blade. The resulting section is stained, placed on a glass slide, and then a drop of glycerin is applied and covered with a coverslip. To detect starch, a solution of iodine in potassium iodide is used: 0.5 g of potassium iodide is dissolved in a small amount of water, 1 g of crystalline iodine is added and water is added to 100 cm 3. A few drops of reagent are applied to a thin piece of sausage, cheese and other material located on a glass slide, the starch turns blue-violet.
Total or Whole Preparations examined without obtaining a section of a tissue or organ. For example, a film of subcutaneous tissue or a crushed preparation of a plant root after fixation, washing and staining is enclosed between a slide and a coverslip. To identify individual structural elements, fixed and stained pieces of subcutaneous tissue or smooth muscle tissue are plucked with a needle on a glass slide - such preparations are called plucked. In some cases, for example, when examining the film of the retina of the eyeball or the skin of a tadpole, after fixation and washing, coloring is not performed, since the cells contain organic inclusions (pigment) that have a natural color.
Method of preparing a histological preparation. The main method for studying fixed cells and tissues is histological, i.e. study of a stained tissue section enclosed in a special medium. To obtain sections, pouring the test material into paraffin or celloidin is used, which makes it possible to obtain thin sections (5-7 microns or 10-30 microns, respectively), for quick preparation of sections (40-60 microns), freezing technique is used.
The main stages in the preparation of a histological specimen are: sampling, fixation, washing, dehydration, embedding in paraffin or celloidin, staining, placing under a coverslip.
Sample selection is made taking into account the purpose of the study and the structure of the material. The size of the sample should not exceed 2.5-3.0 cm on average. Liver and spleen samples are taken with a capsule. Pieces of the atrium are cut out from the heart, since the membranes are thinner than in the ventricles and on one preparation one can see the topographic relationship of the epicardium, myocardium and endocardium. From the kidneys, lymph nodes, adrenal glands, sections of organs are cut out that go deep into the surface perpendicular to the surface so that the cortex and medulla are revealed on the preparation. If it is necessary to prepare preparations of altered organs, pieces of the test material are cut out at the border with the affected area so that the transition zone of the lesion is included in the sample. Samples from skeletal muscles should be cut so that the muscle fibers are in longitudinal and transverse sections. Samples of samples of minced meat, cottage cheese and other crumbling samples are first placed in a piece of gauze and tied with a thread. It should be borne in mind that when opening the chest cavity, the lungs collapse due to elasticity, significantly losing airiness, therefore, a glass or rubber tube is inserted into the trachea of the extracted respiratory organs, through which air is moderately injected. A silk ligature is applied to the trachea below the inserted tube, and a load is tied for complete immersion. To fix the intestines, a section of the organ intended for fixation is preliminarily bandaged with ligatures on both sides. Samples are provided with thick paper labels indicating the number and date of sampling.
fixation, those. preservation of natural (lifetime) architectonics is carried out in order to transfer the living protoplasm of structures to an unchanging state. The action of fixatives is manifested in the fact that as a result of biophysical processes, irreversible coagulation of proteins occurs in tissues and organs. The tissue sample taken from the organ is immersed in the fixative as soon as possible; the ratio of the volume of the material and the fixing liquid must be at least 1:9 (Fig. 3).
Fixation leads to some compaction and a decrease in the volume of the sample. Most often, 10-12% formalin, ethyl alcohol, acetone are used for fixation. To prepare the indicated concentration of the fixing fluid, 40% formalin is diluted with tap water. Ethyl alcohol (100% absolute, or 96%) is used in cases where it is necessary to identify substances that dissolve in aqueous fixatives (for example, glycogen) in sections. In acetone, the material under study (for example, the brain) is fixed for only a few hours. When the organ under study, such as bone tissue, contains lime, decalcification or calcification is carried out. To do this, a small piece of bone is lowered (it is better to hang it on a thread) in a 5-8% aqueous solution of nitric acid, which is changed 2-3 times a day. The result of decalcification is judged by sticking a thin needle into the bone: if there is no resistance, decalcification is completed. After such a pro-
flushing is carried out to remove an excess amount of fixative, for example, after fixation with formalin, washing is carried out with running tap water.
Dehydration carried out to compact the material in ethyl alcohol of increasing concentration: 50-70-100. To prepare 50% and 70% alcohol, 96% alcohol is diluted with distilled water. To prepare 100% alcohol, it is necessary to heat copper sulfate until completely dehydrated and add 96% ethyl alcohol to the dehydrated white powder. In each concentration of alcohol, the material is kept for 1-24 hours, depending on the size of the pieces, the structure of the organ; in 70% alcohol, the material can be kept for several days.
fill is carried out in such a medium that the test sample becomes solid, which makes it possible to obtain thin sections. To do this, use paraffin (impregnation for 1-4 hours), celloidin (impregnation for three weeks). When revealing fats and for the study of loose tissues and organs, pouring into gelatin is used.
slices receive on sledge or rotational microtomes. The thinnest sections (5-7 microns) can be made from a material embedded in paraffin. Sections of 15-20 microns thick are prepared from the material filled with celloidin. To study the microstructure of raw materials and products of animal origin, as a rule, a freezing technique is used. This speeds up the preparation of the preparation, since long stages of dehydration and pouring are eliminated. After fixation and washing, the pieces of the object are placed on a wet stage of a freezing microtome and sections are obtained with a thickness of 40-60 μm. The sections are transferred with a brush into the water, where they straighten out, acquiring the appearance of the thinnest grayish shreds.
Section staining carried out to increase the contrast of various structures in preparations intended for examination in a light microscope. In the process of staining, complex chemical and physical processes occur, therefore, when choosing a method, the selective affinity of cell structures for certain dyes with different physicochemical properties is taken into account.
There are dyes basic (basal), acidic and special. Structures stained with basic dyes are called basophilic, acid dyes - oxyphilic, acidophilic, eosinophilic.
Of the basic (basal) dyes, hematoxylin is used, which stains the chromatin of the nucleus and other structures containing protein in blue or purple; carmine, coloring the core in light red, safranin - dark red paint, thionin - blue paint.
Of the acidic dyes, eosin is used (stains the cytoplasm pink), picric acid (yellow), fuchsin (brick color), indigo carmine (blue).
When studying the microstructure of raw materials and livestock products, combined staining with hematoxylin and eosin is most often used. To do this, sections from water are transferred for 1-3 minutes to a hematoxylin solution; Washed in water for 20-30 minutes, placed in eosin solution for 3-5 minutes and washed with water for 3-5 minutes. The remaining water is removed with filter paper, a drop of 96% ethanol is applied for 1-2 minutes, and dehydrated in a 25% solution of carbolic acid in xylene or toluene. Then 1-2 drops of balm are applied to the cut and covered with a coverslip.
Of the dyes that color fatty inclusions, Sudan 111 is often used. To prepare a dye solution in 95 cm 3 of 85% ethyl alcohol, add 5 cm 3 of acetone and pour Sudan III powder until a saturated solution is obtained (the dye must be contained in excess). The mixture is heated to 50% C and filtered. Sections obtained on a freezing microtome are placed in 50% ethanol for 1-2 minutes, and then in Sudan III for 25 minutes. Sections are rinsed in 50% alcohol, washed with distilled water and embedded in glycerol-gelatin.
Drops of neutral fat turn orange due to the dissolution of Sudan III in fats. Fat inclusions can also be detected with osmic acid, while the fatty structures are stained black.
Conclusion under the coverslip carried out in environments that do not allow air to pass through and are able to keep the cut for a long time. Stained sections are dehydrated in alcohols of increasing strength and placed under a coverslip in fir, Canadian balsam or glycerol-gelatin (the preparation should not come into contact with alcohols and xylene).
Preparation of specimens for electron microscopy. To study biological objects, two types of electron microscopes are used: transmission (transmission) and scanning (raster).
The principle of processing an object for examination in a transmission electron microscope is based on the same principles as for optical microscopes: sampling, fixation, washing, dehydration, pouring, preparation of ultrathin sections, contrasting.
Sample selection is performed taking into account the purpose of the study and the structure of the material, in compliance with the same rules as for light microscopy. The volume of pieces of the studied material should not exceed 1 mm 3 . The main purpose of fixation is to keep the tissue as close to life as possible.
Fixation It is carried out for better preservation of structures, therefore, it is necessary to use reagents having a certain pH and isotonicity, the values of which are determined by the type of tissue being fixed. The fixation process is carried out in two stages: prefixation and postfixation. For prefixation, a solution of 2.5-3% solution of glutaraldehyde (pH 7.2-7.4) is used, the duration of fixation is 2-4 hours. Postfixation is carried out in a 2% solution (pH 7.2-7.4 ) - 1-2 hours. To preserve the intravital structure, it is recommended to use a low-temperature fixative (0-4 °C), and prepare fixatives on phosphate-buffer, cacodylate, veronal-acetate solutions.
flushing it is carried out with 30% cold alcohol 5-7 times, the object is kept in each portion of alcohol for 30 minutes, i.e. washed from the fixative to such a state when the oxidizing action of the fixative ceases.
Dehydration carry out with ethyl alcohols of increasing strength: 30, 50, 70, 96, 100% for 30 minutes. For some pouring methods, the addition of acetone and propylene oxide is recommended for dewatering.
fill carried out in epoxy resins (araldite, epon, etc.). Polymerization of resins in capsules is carried out in a thermostat at 60 °C until hardening, as a rule, within 1-2 days.
Ultrathin sections are obtained using glass knives on an ultramicrotome; for this, epoxy blocks are sharpened in the form of a tetrahedral truncated pyramid. Serial grayish sections are placed in a bath with 10% ethanol. The resulting sections are mounted on copper or palladium meshes, on the surface of which a formvar film is preliminarily applied. To reveal the necessary structures, sections on the meshes are treated with solutions of lead citrate and uranyl acetate.
For scanning electron microscopy the test sample is fixed, dehydrated, depending on the purpose of the study, using the above fixatives. After dehydration, the sample is glued to a holder object, placed in a sputtering unit and coated with a very thin layer of gold or platinum. Unlike transmission electron microscopy, scanning electron microscopy can use corrosive preparations: the object of study is poured with some hardening substances, and then casts and surfaces are examined. They also study replicas obtained by freezing - chipping. In this case, an impression of the cleavage of the surface of the object is examined. To increase the contrast, the replicas must be shaded by spraying metal particles (gold, platinum) or coal.
test questions
- 1. What methods are used in the study of cell structures, tissues and organs?
- 2. What are the basic rules to follow when taking samples for the preparation of histological preparations?
- 3. What are the features of the preparation of preparations from bone tissue?
- 4. How is the test material fixed?
- 5. What is used for dehydration of preparations?
- 6. What media are used to pour the test material?
- 7. What dye is used to detect adipose tissue?
- 8. What is the most commonly used sectioning method and why?
- 9. Name the main stages of preparation of specimens for transmission and scanning electron microscopy.
Histology - ("gistos" in Greek - tissue, logis - teaching) This is the science of the structure, development and vital activity of tissues of multicellular organisms and humans. The objects that are the subject of this science are inaccessible to the naked eye. Therefore, the history of histology is closely connected with the history of the creation of such instruments that allow us to study the smallest objects with the naked eye. 2
The course of histology is conventionally divided into the following sections: n 1. Cytology is the science of the cell. n 2. Embryology is the science of development, from inception to the complete formation of an organism. n 3. General histology - the science of the general patterns inherent in tissues. n 4. Private histology - studies the structure, development of organs and systems.
CYTOLOGY - (Greek κύτος "cell" and λόγος - "study", "science") n A branch of biology that studies living cells, their organelles, their structure, functioning, processes of cell reproduction, aging and death. four
EMBRYOLOGY n (from other -Greek ἔμβρυον - embryo, embryo + -λογία from λόγος - teaching) is a science that studies the development of the embryo. 5
The history of the creation of the cell theory 1590. Jansen invented the microscope, in which magnification was provided by the connection of two lenses. 1665. Robert Hooke first used the term cell. 1650-1700 years. Anthony van Leeuwenhoek first described bacteria and other microorganisms. 1700 -1800 years. Many new descriptions and drawings of various tissues, mainly vegetable, have been published. In 1827 Karl Baer discovered the egg in mammals. 1831 -1833 years. Robert Brown described the nucleus in plant cells. 1838 -1839 years. The botanist Matthias Schleiden and the zoologist Theodor Schwann combined the ideas of different scientists and formulated the cell theory, which postulated that the basic unit of structure and function in living organisms is the cell. 1855 Rudolf Virchow showed that all cells are formed as a result of cell divisions.
The history of the creation of the cell theory 1665. Examining a section of cork under a microscope, an English scientist, physicist Robert Hooke discovered that it consists of cells separated by partitions. These cells he called "cells"
The history of the creation of the cellular theory In the 17th century, Leeuwenhoek designed a microscope and opened the door to the microcosm for people. A variety of ciliates, rotifers and other tiny living creatures flashed before the eyes of the astonished researchers. It turned out that they are everywhere - these smallest organisms: in water, manure, in air and dust, in earth and gutters, in rotting waste of animal and vegetable origin.
The history of the creation of the cell theory 1831-1833. Robert Brown described the nucleus in plant cells. In 1838, the German botanist M. Schleiden drew attention to the nucleus and considered it to be the originator of the cell. According to Schleiden, a nucleolus condenses from a granular substance, around which a nucleus is formed, and around the nucleus - a cell, and the nucleus may disappear in the process of cell formation.
The history of the creation of the cellular theory The German zoologist T. Schwann showed that animal tissues also consist of cells. He created a theory stating that cells containing nuclei represent the structural and functional basis of all living things. The cellular theory of structure was formulated and published by T. Schwann in 1839. Its essence can be expressed in the following provisions: 1. The cell is the elementary structural unit of the structure of all living beings; 2. Cells of plants and animals are independent, homologous to each other in origin and structure. Each cell functions independently of the others, but together with all. 3. All cells arise from structureless intercellular substance. (Mistake!) 4. The life activity of the cell is determined by the shell. (Error!)
The history of the creation of the cell theory In 1855, the German physician R. Virchow made a generalization: a cell can only arise from a previous cell. This led to the realization of the fact that the growth and development of organisms are associated with cell division and their further differentiation, leading to the formation of tissues and organs.
The history of the creation of the cell theory by Karl Baer Back in 1827, Karl Baer discovered the egg in mammals, proved that the development of mammals begins with a fertilized egg. This means that the development of any organism begins with one fertilized egg, the cell is the unit of development.
The history of the creation of the cellular theory 1865 The laws of heredity were published (G. Mendel). 1868 Nucleic acids were discovered (F. Miescher) 1873 Chromosomes were discovered (F. Schneider) 1874 Mitosis was discovered in plant cells (I. D. Chistyakov) 1878 Mitotic division of animal cells was discovered (W. Fleming, P. I. Peremezhko) 1879 Fleming - the behavior of chromosomes during division. 1882 Meiosis was discovered in animal cells (W. Fleming) 1883 It was shown that the number of chromosomes in germ cells is two times less than in somatic cells (E. Van Beneden) 1887 Meiosis was discovered in plant cells (E. Strasburger ) 1898 Golgi discovered the mesh apparatus of the cell, the Golgi apparatus. 1914 The chromosome theory of heredity was formulated (T. Morgan). 1924 The natural-scientific theory of the origin of life on Earth was published (A. I. Oparin). 1953 Ideas about the structure of DNA were formulated and its model was created (D. Watson and F. Crick). 1961 The nature and properties of the genetic code are determined (F. Crick, L. Barnet, S. Benner).
The main provisions of modern cellular theory 1. A cell is an elementary living system, a unit of structure, vital activity, reproduction and individual development of organisms. 2. The cells of all living organisms are homologous, uniform in structure and origin. 3. Cell formation. New cells arise only by dividing pre-existing cells. 4. Cell and organism. A cell can be an independent organism (prokaryotes and unicellular eukaryotes). All multicellular organisms are made up of cells. 5. Functions of cells. In cells, metabolism, irritability and excitability, movement, reproduction and differentiation are carried out. 6. Cell evolution. The cellular organization arose at the dawn of life and went a long way of evolutionary development from nuclear-free forms (prokaryotes) to nuclear forms (eukaryotes).
METHODS FOR MICROSCOPY OF HISTOLOGICAL SPECIMENS 1. Light microscopy. 2. Ultraviolet microscopy. 3. Fluorescent (luminescent) microscopy. 4. Phase contrast microscopy. 5. Dark field microscopy. 6. Interference microscopy 7. Polarization microscopy. 8. Electron microscopy. 17
Microscope n This optical instrument allows you to observe small objects. Image magnification is achieved by a system of objective lenses and an eyepiece. The mirror, condenser and diaphragm direct the light flux and regulate the illumination of the object. The mechanical part of the microscope includes: a tripod, an object table, macro- and micrometer screws, a tube holder. eighteen
Special methods of microscopy: - phase-contrast microscope - (for the study of live non-stained objects) - microscopy allows you to study live and unstained objects. When light passes through colored objects, the amplitude of the light wave changes, and when light passes through uncolored objects, the phase of the light wave changes, which is used to obtain a high-contrast image in phase-contrast and interference microscopy. - dark-field microscope (for studying living unstained objects). A special condenser is used that highlights the contrasting structures of the unpainted material. Dark-field microscopy makes it possible to observe living objects. The observed object appears as illuminated in a dark field. In this case, the rays from the illuminator fall on the object from the side, and only scattered rays enter the microscope lenses. 19
Special methods of microscopy Luminescent mic-p (for the study of living unstained objects) Microscopy is used to observe fluorescent (luminescent) objects. In a fluorescent microscope, light from a powerful source passes through two filters. One filter blocks light in front of the sample and allows light of the wavelength that excites the sample to fluoresce. The other filter allows light of the wavelength emitted by the fluorescent object to pass through. Thus, fluorescent objects absorb light of one wavelength and emit light in another region of the spectrum. -ultraviolet ability m-pa) mic-p (increases the resolution -polarization mic-p (for research objects with an orderly arrangement of molecules - skeleton, muscle, collagen fibers, etc.) microscopy - image formation of uncolored anisotropic structures ( such as collagen fibers and myofibrils).20
Special methods of microscopy - interference microscopy (to determine the dry residue in cells, determine the thickness of objects) - microscopy combines the principles of phase-contrast and polarization microscopy and is used to obtain a contrast image of unstained objects. Special interference optics (Nomarsky optics) have found application in microscopes with differential interference contrast. C. Electron microscopy: -transmission (study of objects through transmission) -scanning (study of the surface of objects) Theoretically, the resolution of a transmission EM is 0.002 nm. The real resolution of modern microscopes approaches 0.1 nm. For biological objects, the EM resolution in practice is 2 nm. 21
Special Microscopy Techniques A transmission electron microscope consists of a column through which electrons emitted by a cathode filament pass in vacuum. An electron beam focused by ring magnets passes through the prepared sample. The character of electron scattering depends on the density of the sample. Electrons passing through the sample are focused, observed on a fluorescent screen, and recorded using a photographic plate. A scanning electron microscope is used to obtain a three-dimensional image of the surface of the object under study. The chipping method (freezing-cleaving) is used to study the internal structure of cell membranes. Cells are frozen at liquid nitrogen temperature in the presence of a cryoprotectant and used to make chips. The cleavage planes pass through the hydrophobic middle of the lipid bilayer. The exposed inner surface of the membranes is shaded with platinum, the resulting replicas are studied in a scanning EM. 22
Special (non-microscopic) methods: 1. Cyto- or histochemistry - the essence is the use of strictly specific chemical reactions with a light end product in cells and tissues to determine the amount of various substances (proteins, enzymes, fats, carbohydrates, etc.). Can be applied at the level of a light or electron microscope. 2. Cytophotometry - the method is used in combination with 1 and makes it possible to quantify proteins, enzymes, etc. identified by the cytohistochemical method. 3. Autoradiography - substances containing radioactive isotopes of chemical elements are introduced into the body. These substances are included in metabolic processes in cells. Localization, further movement of these substances in the organs are determined on histological preparations by radiation, which is captured by a photographic emulsion applied to the preparation. 4. X-ray diffraction analysis - allows you to determine the amount of chemical elements in cells, to study the molecular structure of biological micro-objects. 24 5. Morphometry - measuring the size of biol. structures at the cellular and subcellular levels.
Special (non-microscopic) methods 6. Microurgy - carrying out very delicate operations with a micromanipulator under a microscope (nucleus transplantation, introduction of various substances into cells, measurement of biopotentials, etc.) 6. Method of culturing cells and tissues - in nutrient media or in diffusion chambers, implanted in various body tissues. 7. Ultracentrifugation - fractionation of cells or subcellular structures by centrifugation in solutions of various densities. 8. Experimental method. 9. Method of tissue and organ transplantation. 25
Fixation preserves the structure of cells, tissues and organs, prevents their bacterial contamination and enzymatic digestion, and stabilizes macromolecules through their chemical crosslinking. 32
Fixing liquid formalin, alcohols, glutaraldehyde - The most common fixatives; Cryofixation - The best preservation of structures is ensured by instantaneous freezing of samples in liquid nitrogen (-196 ° C); Lyophilization - small pieces of tissue are subjected to rapid freezing, which stops metabolic processes. Dehydration - the standard procedure for removing water is dehydration in alcohols of increasing strength (from 70 to 60%). Filling - makes the fabric durable, prevents it from crushing and wrinkling during cutting, makes it possible to obtain cuts of standard thickness. The most common embedding medium is paraffin. Celloidin, plastic media and resins are also used. 33
Dehydration prepares the fixed tissue for penetration of the embedding media. Water from living tissue, as well as water from fixing mixtures (most fixatives are aqueous solutions) must be completely removed after fixation. The standard procedure for removing water is dehydration in alcohols increasing from 60° to 100° strength. 34
Filling is a necessary procedure that precedes the preparation of sections. Filling makes the fabric durable, prevents it from being crushed and wrinkled during cutting, and makes it possible to obtain thin sections of standard thickness. The most common embedding medium is paraffin. Celloidin, plastic media and resins are also used. 35
Rotary microtome. 40 n Blocks containing a piece of an organ are fixed in a movable object holder. When it is lowered, serial sections remain on the knife, they are removed from the knife and mounted on a glass slide for further processing and microscopy.
Histosection staining methods: n Nuclear (basic): n hematoxylin - stains n n n n nuclei blue; iron hematoxylin; azur II (in purple); carmine (in red); safranin (in red); methyl blue (to blue); toluidine (in blue); thionine (in blue). n Cytoplasmic- (acid): n eosin - in pink; n erythrosin; n orange "G" ; n sour fuchsin - to red; n picric acid - yellow; n Congo - red - to red 44
SPECIAL Methods for staining histosections n Sudan III – orange staining of lipids and fats; n osmic acid - coloring of lipids and fats in black; n orcein - brown coloring of elastic fibers; n silver nitrate - impregnation of nerve elements in a dark brown color. 45
Cell structures: n OXYPHILIAn the ability to stain pink with acidic dyes n Basophilian the ability to stain blue with basic dyes n Neutrophilia - n the ability to stain purple with acidic and basic dyes. 47
1
Cell n is an elementary living system consisting of cytoplasm, nucleus, membrane and is the basis for the development, structure and life of animal and plant organisms.
The glycocalyx is an epimembrane complex composed of protein-bound saccharides and lipid-bound saccharides. Functions n Reception (hormones, cytokines, mediators and antigens) n Intercellular interactions (irritability and recognition) n Parietal digestion (microvilli of intestinal border cells)
Functions of the cytolemma: - delimiting; - active and passive transport of substances in both directions; - receptor functions; contact with neighboring cells.
Main Research objects are histological preparations, and the main research method is microscopy.
The histological preparation should be sufficiently transparent (thin) and contrast. It is made from both living and dead (fixed) structures. The preparation can be a suspension of cells, a smear, an imprint, a film, a total preparation and a thin section.
The process of making histological preparations for microscopic studies includes the following main steps: 1) taking the material and fixing it; 2) material compaction; 3) preparation of sections; 4) staining, or contrasting sections; 5) conclusion of sections.
For staining, special histological dyes are used with different pH values: acidic, neutral and basic. The structures stained by them are called oxyphilic, neutrophilic (heterophilic) and basophilic, respectively.
What methods does histological science use? They are quite numerous and varied:
Microscopy.
Light microscopy. Modern microscopes have high resolution. Resolution is defined as the smallest distance (d) between two adjacent points that can be seen separately. This distance depends on the light wavelength (λ) and is expressed by the formula: d = 1/2 λ.
The minimum wavelength of the visible part of the spectrum is 0.4 µm. Therefore, the resolving power of the light microscope is 0.2 µm, and the total magnification reaches 2500 times.
ultraviolet microscopy . The wavelength of ultraviolet light is 0.2 µm, therefore, the resolution of the ultraviolet microscope is 0.1 µm, but since ultraviolet radiation is invisible, a luminescent screen is needed to observe the object under study.
Fluorescent (luminescent) microscopy. Short-wave (invisible) radiation, being absorbed by a number of substances, excites their electrons, which emit light with a longer wavelength, becoming the visible part of the spectrum. Thus, an increase in the resolution of the microscope is achieved.
Phase contrast microscopy allows you to emit uncolored objects.
Polarizing microscopy used to study the architectonics of histological structures, such as collagen fibers.
electron microscopy makes it possible to study objects magnified tens of thousands of times.
Microphotography and microfilmography . These methods make it possible to study fixed objects in photographs and living microscopic objects in motion.
Methods of qualitative and quantitative research.
Histo –and cytochemistry , including quantitative, allows for a qualitative analysis of the objects under study at the tissue, cellular and subcellular levels.
Cytospectrophotometry It makes it possible to study the quantitative content of certain biological substances in cells and tissues based on the absorption of light of a certain wavelength by the dye associated with them.
Differential centrifugation allows you to separate the contents of cells that differ from each other in their mass.
Radiography It is based on the inclusion of a radioactive label (for example, radioactive iodine, H³-thymidine, etc.) in the metabolic process.
Morphometry allows you to measure the areas and volumes of cells, their nuclei and organelles using eyepiece - and object-micrometers and special grids.
Computer application for automatic processing of digital material.
Tissue culture method is the maintenance of viability and division of cells and tissues outside the body. For this, special containers with a nutrient medium are used, in which all the necessary conditions for the vital activity of cells are created. Using this method, it is possible to study the differentiation and functional development of cells, the patterns of their malignant transformation and the development of a tumor process, intercellular interaction, damage to cells and tissues by viruses and microorganisms, the effect of drugs on metabolic processes in cells and tissues, etc.
Intravital (vital) staining used to study the phenomena of phagocytosis and the activity of macrophages, the filtration capacity of the renal tubules, etc.
Tissue transplantation method. This method is used to study the behavior of cells and their morphofunctional state when they are transplanted into another organism. For example, this method is used to keep animals exposed to lethal doses of radiation.
Micromanipulation. This method has been used in molecular biology, genetic engineering, and also in cloning, when a nucleus is removed from an egg with a haploid set of chromosomes using a micromanipulator and a somatic cell nucleus with a diploid set of chromosomes is transplanted into it.
Exists many research methods, among which the following stand out: observation of living embryos using film and video recording (used mainly in the experiment). For this, a special microphotographic device is used, which is connected to a thermal chamber in which the embryo develops. When studying the development of a chicken embryo, for example, you make a window in the shell, which is closed with a transparent plate. This method made it possible to trace and refine the dynamics of changes in the shape and size of embryos in the process of development.
Fixed Slice Method embryos using light and electron microscopy, historadioautography, histo- and immunocytochemistry. These methods make it possible to analyze tissue and intracellular changes in the dynamics of development of parts of the embryo. With the help of histo- and immunocytochemical methods, biochemical processes occurring in embryonic cells are studied - the synthesis of DNA, RNA, proteins, specific receptor proteins, etc. Using these methods, important information was obtained on cell and tissue differentiation in the development of embryos and fetuses.
Marking method, proposed in 1925 by W. Vogt (1888-1941), makes it possible to study cell movements in a developing embryo. For this, markers that are non-toxic to the embryos (for example, neutral red, charcoal particles), as well as antibodies to certain proteins, are used. When using antibodies, their ability to combine with a fluorescent dye and germ proteins is used. With the help of fluorescent microscopy, the distribution of the dye is traced and the dynamics of protein synthesis in the developing tissues of the embryo is studied.
Microsurgery methods were developed at the beginning of the 20th century by representatives of the school of G. Spemann (1869-1941). They included: removal of the shells of animal eggs, transplantation of parts of one embryo to another, etc. These methods are also used to study the consequences of destruction (for example, using a laser beam) of parts of the embryo or its individual cells. Transplantation as a kind of microsurgery is used to identify the pathways of cell migration and sources of tissue development. At the same time, a site of the embryo, for example, a quail, is transplanted into the same site of the chicken embryo in place of the remote site. Quail cell nuclei have a characteristic structure and are therefore distinguishable from the nuclei of chicken embryo cells.
explantation- excision of a small area of the embryo and its cultivation in an artificial environment. Using this method, it is possible to obtain information about the sources of tissue development from a given area of the embryo and to identify histogenetic patterns of development.
Nuclear transplant- a method for cloning embryos. For example, the transplantation of nuclei from the cells of the intestinal epithelium of the clawed frog tadpole into a frog egg, the nucleus of which was inactivated by an ultraviolet ray, led to the appearance of new individuals (Gerdon's experiments). These experiments laid the foundation for the cloning of higher vertebrates and contributed to the emergence (in 1997) of the famous sheep Dolly. Similar embryological experiments have convincingly shown that the nuclei of somatic cells contain a complete set of genetic information for the development of a new organism.
The latest achievement experimental embryology was the development of the method of in vitro fertilization. The transplantation of embryos conceived in a test tube into the uterus is the basis of infertility treatment. In 1973, L. Shettles (USA) extracted a preovulatory egg from the ovary of an infertile woman and fertilized it with her husband's spermatozoa. This was the beginning of the technique of transplanting human embryos to treat infertility. However, only in 1978 in the UK, as a result of a successful transplantation into the uterus of an infertile woman of a human embryo at the stage of 8 blastomeres, after 2.5 days of cultivation, the world's first "test-tube" child weighing 2700 g appeared.