PRACTICUM IN HISTOLOGY, CYTOLOGY, AND EMBRYOLOGY - 2016

Chapter 1. HISTOLOGICAL TECHNIQUE.

Histology is the science that studies the Tissues and Cells of Organs and systems in Multicellular Organisms. Histology consists of several distinct branches. The Study of The Cell is known as Cytology. General Histology examines tissue Structure, whereas Special Histology focuses on The structure of the body's organs and systems. In the histology course, Cells and Tissues are studied in the context of their origin and development; consequently, Embryology, as the science of embryonic development, forms an integral part of histology.

Cytology, histology, and embryology employ specialized Research Methods collectively referred to as histological techniques. All of these methods require The Use of a Microscope and are therefore classified as microscopic methods.

The MAIN STAGES OF histological analysis include selecting the specimen, preparing it for study, microscopic examination, Qualitative and quantitative analysis, and recording the results.

The Objects of Study may be living or dead (fixed) cells and tissues. The Study of Living tissues and cells is carried out using vital or supravital methods, while fixed specimens require post-vital methods. The latter necessitate the preparation of permanent histological specimens (microscopic slides). Histological slides refer to stained sections, films, smears, imprints, or isolated cells prepared from tissues (organs), placed on a Glass microscope slide, embedded in a thin layer of a transparent medium, and covered with a coverslip (Fig. 1). The main stages of preparing a histological slide include: obtaining the material, fixation, washing, dehydration, embedding, mounting, and sectioning and staining.

Obtaining the material. The material—small pieces of tissue or organ—is excised using sharp scissors or a scalpel in a way that avoids unnecessary trauma. The volume of the tissue blocks is approximately 0.5 — 2.0 cm³. The material must be fresh, meaning it should be collected as soon as possible after the death of a human or experimental animal.

Fixation of the material. The collected tissue block is immersed in a fixing fluid, or fixative. The volume of the fixative should exceed the volume of the tissue block by 50 to 100 times. Fixatives can be simple or compound. The most common simple fixative is a 10% formalin solution. Ethyl and methyl alcohols, solutions of heavy metal salts, as well as acetic, picric, and osmium acids, among others, are also used. Compound fixatives are mixtures composed of individual components in specific ratios. Examples include Lillie's fluid (96° alcohol, formalin, glacial acetic acid), Carnoy's fluid (absolute alcohol, chloroform, glacial acetic acid), and others.

Class="center">

Fig. 1. Histological specimens.

A - stained section on a microscope slide.

B - the same section covered with a coverslip.

Fixation can be performed at room Temperature, or at a reduced temperature when utilizing histochemical research methods. The duration of fixation depends on The properties of the fixative and the size of the specimen, ranging from a few minutes to several days.

The choice of fixative is determined by the objective of fixation—namely, preserving the in vivo state of the morphological structures of the tissue or organ at the moment of the Organism's death with minimal artifacts caused by the fixation process. Fixation facilitates the subsequent uptake of stains by the specimens and prevents putrefactive processes.

Washing the material. Following fixation, the material is typically washed in running tap Water for 24 to 48 hours to remove residual fixative.

Dehydration and embedding of the material. The purpose of these stages is to remove water from the tissue blocks and prepare them for sectioning into thin slices.

Dehydration of tissue blocks fixed In aqueous solutions is carried out gradually using a series of alcohols of increasing concentrations: 50^o, 60^o, 70^o, 80^o, 100^o (absolute alcohol). Absolute alcohol is obtained from 96^o alcohol by dehydration with copper sulfate or by distillation over anhydrous copper sulfate or molecular sieves. The duration of immersion in alcohols of various concentrations depends on the specimen and its size (ranging from a few hours to several days).

Embedding is achieved by impregnating the tissue blocks with liquid media that subsequently solidify (such as paraffin or celloidin). Since these substances are soluble in xylene,

benzene, or toluene, the tissue blocks are first immersed in a 1:1 mixture of alcohol and xylene, then through two to three changes of pure xylene, and subsequently into a mixture of xylene and paraffin at 55°C - 56°C. Metal, paper, or ceramic Molds are used for embedding the material and forming blocks.

Sectioning. Thin sections (5 - 7 µm thick) can be cut from paraffin or other blocks using a specialized device called a microtome. Microtome knives are used for this purpose. The sections are then collected and mounted on microscope slides previously coated with a 1:1 mixture of egg albumin and glycerin.

Sections for histochemical studies can be prepared using a freezing microtome or a cryostat microtome. A cryostat microtome is a freon-cooled refrigeration unit with a built-in microtome housed inside its chamber. The working temperature within the chamber (ranging from 0^oC to - 20^oC) is maintained automatically. The cryostat allows for The production of thin sections from unfixed tissue.

Thin sections are transparent; therefore, to distinguish the structural details of a tissue or organ, they must be stained.

Staining of sections. There are numerous staining methods, utilizing over three thousand different Dyes. Histological dyes can be broadly classified into general and special; according to their origin, into plant, animal, and synthetic; and according to their chemical properties, into basic, acidic, and neutral.

General stains are used to study the General Morphology of cells, tissues, and organs. Nuclear stains include hematoxylin, carmine, azure II, and safranin. Hematoxylin is derived from the bark of the logwood tree (Haematoxylum campechianum), which grows in South America. It stains cell nuclei a blue-violet color. Carmine is an extract derived from cochineal scale insects and stains nuclei bright red; safranin stains them dark red, and azure II stains them purple. These are basic cationic dyes containing positively charged nitrogen atoms. Histological structures that bind basic stains are termed basophilic.

Cytoplasmic stains—such as eosin, erythrosin, and acid fuchsin—color the Cytoplasm in various shades, most commonly in different tones of red. Chemically, they are acidic or anionic dyes, and by origin, they are synthetic. Structures that readily bind acidic stains are referred to as oxyphilic (acidophilic, eosinophilic).

Neutral dyes are dye mixtures containing both acidic and basic components.

Special stains selectively color specific substances or structures. For example, Sudan III stains Lipids orange, orcein stains elastic fibers brown, and so on.

Histological structures can also be visualized using the impregnation method. This technique involves treating tissue blocks or sections with heavy metal salt solutions followed by their reduction.

The section Staining Procedure begins with deparaffinization—Treatment with xylene or toluene—followed by rehydration through a descending alcohol series. Next, following the protocol specific to each method, the sections are treated sequentially with dye solutions. After staining, the sections are dehydrated through an ascending alcohol series and mounted in a transparent medium, such as Canada balsam or polystyrene, which have refractive indices similar to that of glass.

Histological slides prepared in compliance with the aforementioned requirements can be preserved for a very long time.