Living tissues are generally trickier to handle and are important for short period only. In living cell, structure and function may be studied concurrently. Living cells may be observed to move to ingest foreign material, infrequently to divide and to perform other functions. Dead or fixed cells can't do these things.
Observing Living Tissue:
Free cells are colorless and structures within them lack contrast. This complexity may be beat by using phase contrast microscope like web of frog foot, the wing of bat and the buccal pouch of the hamster. Minute pieces of tissue for microscopic examination may be removed placed in some fairly harmless liquid like serum or a 0.85 per cent aqueous solution of sodium chloride, and teased separately quietly with needles of fine steel or glass.
Prolonged preservation of living cells outside the body can be achieved through tissue culture: fragments of tissue are detached aseptically, transferred to physiological medium and kept at temperature normal for animal from which tissue was taken. Tissue culture is important method for study of cancer and of several viruses. Microdissection involves use of the instrument that moves very fine glass needles with precision under the microscope.
Preparation of Dead Tissue:
1) Light Microscopy:
Use is composed of sections that are thin slices cut from piece of fixed tissue that is then stained, mounted in medium of appropriate refractive index on slide, and at last covered with covership. Procedure for producing histological section involves the following steps:
a) Removal of Specimen: For cytological purposes and for best histological preparations, material must be removed from anesthetized animal. Surgical material represents source of human material since, often, some normal tissue is removed together with abnormal tissue or diseased tissue.
b) Fixation: Primary objective of fixation is to preserve protoplasm with least alteration form living state. Fixing fluids serve as preservative, inhibiting autolytic changes and bacterial growth. They coagulate protoplasm therefore rendering it insoluble and harden tissue so that sectioning is assisted. They may or may not conserve carbohydrates and lipids. Several fixatives also increase affinity of protoplasm for certain stains.
c) Embedding: Prior to embedding, fixed specimen is washed to eliminate excess fixative and then dehydrated by passing it by increasing strengths of alcohol or some other dehydrating agent. Tissue/specimen is then cleaned; this involves removal of dehydrating agent and its replacement by some fluid which is miscible both with dehydrating agent and embedding medium. After infiltration, embedding agent is made to solidify so that firm homogenous mass having embedded tissue/specimen is obtained.
d) Sectioning: Tissue embedded in paraffin may be cut extremely thin to between 3 and 10 microns (μ) thick using micrometer. Each section is transferred to clean glass microscope slide on which little egg albumen has been smeared.
e) Staining: The purpose of staining is to improve natural contrast and to make more evident different cell and tissue components and extrinsic materials. Most stains are dissolved in aqueous solution that will not mix with paraffin; and therefore to stain paraffin section it is essential to remove paraffin by placing the section in a paraffin solvent or decerating agent, usually xylol or tolud.
f) Mounting: After staining, excess dye is removed by washing with water or alcohol, depending on solvent of dye, and section is dehydrated by ascending grades of alcohol. After removal from clearing agent, drop of mounting medium eg. Canadian balsam, which has a similar refractive index to that of glass, is placed on the section.
2) Electron Microscopy:
Generally, method of preparing biological materials for electron microscopy is like that stated for light microscopy with some significant points of difference. Much smaller pieces of tissue are utilized as preservation and fixation of cell fine structure is more vital and needs rapid interaction with fixative. Blocks are commonly 1 cu. Mm or less in size.
This is another method of making materials for electron microscopy. This method comprises only physical preparation that may permit examination of specimens virtually free of artifacts. Specimen is frozen, cut in small pieces, briefly warmed to etch cut surface by vacuum - sublimation and a replica of the surface made by heavy metal shadowing.
This is special method that uses microscope, either light or electron, only as visual aid. Method involves introducing tracer isotopes in organism either by feeding or by injection; tracers follow same metabolic pathways as do naturally occurring elements. Their presence in organ can be detected by autoradiography. After the administration of a tracer isotope, organ or tissue under investigation is removed and processed in normal manner for light or electron microscopy. This method has been utilized to get some excellent results: for example, localization of radioiodine in thyroid gland and of phosphorous (using radiostrontium) as substitute in bone.
Examination and Interpretation of sections:
The ability to interpret histological section is a skill which you have to build up. In gross anatomy, structure is studied in three dimensions. Though, sections are viewed in two-dimensional frame. It therefore takes good frame of mind for student to relate two dimensional (3-d) form. It is significant to reconstruct the 3-d mental picture of cells, tissues and organs from two-dimensional (2-d) sections.
Dead structures are examined for objective of throwing light on their condition in life. Conditions that were hitherto dynamic in life have been converted to the static form in permanent histological section.
In general dyes utilized for staining are difficult organic chemicals that frequently show some variability in performance. They may be categorized based on their use with regards to tissue or cell components. Dyes may be of common use staining either nucleus or cytoplasm, or they may be specific with regard to specific components. E.g. some may stain for fats, protein, carbohydrates, chloroplasts Golgi body etc. A basophil is stain whose coloring properties is in basic radical of the neutral salt, and in most cases is attracted to substances with acidic tendencies like ribonucleic acid (RNA).
This is the field of research that is expanding quickly and is based on fact that deposition of particular stain in definite regions is result of chemical or physical properties of structure. Histo chemical methods are even now available for several inorganic and organic substances. E.g. Prussian blue stain is utilized for detection of ferric iron; in sections treated with potassium ferrocyanide deposits are colored blue. Feulgen reaction for recognition of deoxyribonucleic acid (DNA) is example of histochemical test.
Not all reactions in Histochemistry depend on chemical affinities. Fat can be detected in section that haven't been exposed to fat solvents by stain like Sudan III, Sudan IV and Sudan black B. Such stains have physical similarity for lipids and are absorbed by fat. Immunocytochemistry is one branch of Histochemistry which is gaining grounds. At light microscopic level, fluorescent antibody method is a sensitive method for localization of particular polysaccharides or protein. Basis for immnocyto-chemistry is the fact that body reacts to foreign substances, antigens, by generating specific substances, antibodies that combine with and inactivate antigens. Method has been utilized to recognize cells of origin of protein hormones, intracellular localization of different enzymes and sites of protein like myosin. Method has been adapted for use with electron microscope by conjugating antibody with metalloprotein like ferritin that naturally has a distinct appearance in electron microscope. This method allows investigator to localize precisely site of antigen antibody reaction.
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