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Microscopy: - Light microscopy: Usually three objectives are used, low power with magnification of 10 times, high power with 45 times and oil immersion 100 times). The shorter the wavelength of the light used the better is the resolution, and with ultraviolet light it can be improved two to three times. Examination of living tissue: Living tissue is transparent and the homogeneity in optical density of its component hides its detailed structures. Staining techniques must therefore be used to see cellular details, but these must be performed on dead fixed tissues. In supravital techniques, e.g. mitochondria of living leukocytes can be stained by Janus green but even this damages them rapidly so that the cells soon lose their motility and begin to die. Three techniques have been developed to overcome these difficulties in examining living cells. - Dark-ground illumination: Dark-ground illumination relies upon the fact that objects placed in a beam of light may be seen by the rays which they reflect. This method demonstrates the organisms which cannot be readily stained, e.g. Treponema pallidum. - Phase contrast Microscopy: Different parts of a cell have different refractive index. These differences are converted into differences in optical density. Living cells are examined by this method. This is commonly used in virology - Interference Microscopy: The light that passes through the microscope is split into two beams. The light passing through the specimen is retarded, and interferes with the light of the other beam when the two are recombined in an image plane. The method finds little application in routine pathology.
Examination of fixed tissue: Paraffin section: It is the most commonly used routine method of examination. The tissue is fixed, dehydrated in graded alcohols, cleared in xylol, chloroform or other solvent which is miscible with both alcohol and wax, and finally embedded in paraffin wax. Common fixative is formaldehyde (10% aqueous solution of formalin). Staining: Hematoxylin and Eosin staining (with its limitation) is preferred for paraffin sections because it is relatively quick, inexpensive and allows accurate diagnosis of the vast majority of cases received in a laboratory. Nucleus takes the colour of hematoxylin (blue) and cytoplasm eosin (pink). In paraffin section certain substances are removed (e.g. fat )and alters other substances (e.g. enzymes and some antigens). Hence frozen sections are used. Frozen section: Frozen section is done in cryostat (microtome in which sections are cut at - 30 C.) This method is used for preservation of fat, enzymes and some antigens (lost in paraffin method). Commonly used stain for neutral fat is Sudan III or oil red O; for other tissues Toluidine blue is used. Histochemistry: For different types of tissues, enzymes and antigens different stains are used. Example: - Van Gieson’s stain is used to differentiate muscle and connective tissue). - Hale's colloidal iron stain (Prussian blue) is used to stain iron. - Periodic acid-Schiff (PAS) is used to stain glycogen, the glycoproteins of ground substance and basement membrane, and epithelial mucin. - Fuelgen stain is used to show nuclei and chromosomes (magenta coloured). - Perl's Prussian-blue reaction is used to stain hemosiderin. - Reduction of silver nitrate to metallic silver to show argentaffin cells (black). Metachromasia means the change of colour of the stain in the coloured tissue or cell (Eg. Toluidine blue stains the granules of mast cells and some acid mucopolysaccharides red ). Immunohistochemistry: The antibody can be labelled with fluorescein and is examined under ultraviolet light. Alternative method: i) Sandwich technique using commercial labelled Coomb’s reagent. ii) Radioactive isotopes in conjunction with autoradiography iii) Immunoperoxide technique. iv) Double staining by using two separate antigen in one section. This is done by using horseradish peroxidase with different substrate chromogen system. Electron microscopy: The tissue must be fixed immediately after removal from the body, commonly in osmium tetroxide. Other fixative is glutaraldehyde. Formaldehyde fixes slowly. It is useful in the morphological diagnosis of poorly differentiated tumours and to differentiate various types of glomerulonephritis and epidermolysis bullosa. Ultracentrifuge: The ultracentrifuge is used to separate particles of different sizes from a mixture. The technique is used mainly to separate macroglobulins. Electrophoresis: If a mixture of proteins is placed in an electric field at a known pH, individual proteins move at particular rates dependent mainly to their size and charge. Chromatography: Chromatography is an important technique for separating pure substances from mixtures. The separation depends on the fact that different substances follow the moving solvent at different rates. Large molecules cannot enter the beads and so pass rapidly through the column. Small particles enter the beads and pass slowly through the column. Chromatography is of great use in the separation and purification of proteins. It is also used for separation of aminoacids or sugars in solution such as urine. Radioactive isotopes: Radioactive isotopes are treated by living cells in the same way as the normal elements. Their radiation can be detected by suitable counter e.g. investigation of thyroid function by radioactive iodine. The isotopes can also be used at a microscopic level. A tissue section or peritoneal spread is placed on a photographic film subsequent photographic development will reveal the site of isotope localization as black grain. An example is seen in mast cell granules in peritoneal spread [International J Appl Radiation & Isotopes (London & New York), 1959]. Tissue culture: Tissue culture is an extremely important technique to study the behavior of cancer cell and normal cell. The technique is used to cultivate viruses and to study the tissue metabolism. Microdissection: [ Microdissection is a technique that is very useful both in the research setting and for clinical molecular testing in paraffin-embedded tissue samples. The available techniques range from simple and inexpensive (manual microdissection) to complex and expensive (laser-capture microdissection). All of the techniques, however, require the user to be familiar with microscopy and histology.] (Arch Pathol Lab Med. 2004;128:1372-1378) A cell can be dissected and its nucleus can be removed. Micro-beams of ultraviolet light, or Laser rays (Light Amplification by stimulated Emission of Radiation), have been used to produce damage in a particular part of a cell. Micromanipulation of embryos can produce allophonic mouse. Genetic alteration can produce clones of different genetic constitution.
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To understand the pathological changes, knowledge of cell structure, physiology and the intercellular connective tissue is essential : Connective tissue: Connective tissue is composed of : 1. Ground substance 2. Cells: fibroblasts, macrophages, mast cells, plasma cells, lymphocytes, eosinophils. 3. Fibers: collagen fibers, reticulin fiber, elastic fiber 4. Blood and lymphatic capillaries To understand the pathological changes, knowledge of cell structure, physiology and the intercellular connective tissue is essential. |
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Legionellosis (Legionnaires' Disease)
Atypical Mycobacterial Infection Mycobacterium Avium Intracellulare Mycobacterium Kansasii Infection Histoplasmosis (Histoplasma Capsulatum) Aetiology and Pathogenesis of Mesothelioma Gross features of Mesothelioma Microscopic features of Mesothelioma Cytological Diagnosis of Mesothelioma Histochemistry and Immuno histochemistry in the diagnosis of Mesothelioma WELL DIFFERENTIATED PAPILLARY MESOTHELIOMA LOCALIZED MALIGNANT MESOTHELIOMA Electron microscopy of Mesothelioma Pseudo-mesotheliomatous Adenocarcinoma Mesothelioma of Atrioventricular Node |