VAN 211: Veterinary Histology and Embryology (2+2)

GENERAL HISTOLOGICAL TECHNIQUE

The technique includes methods of preparation and examination of tissues and cells.

Methods of Preparation

• Dissociation or teasing of tissues: In this method details of individual cells are well seen but their anatomical relationships are destroyed. The organ or tissue is dissociated in some fluid medium and examined under microscope. The fluid medium used usually has the property of dissolving the intracellular cement substance at the same time fixes and preserves the cell structure. (Muller’s fluid, 1% Normal saline, 30% Alcohol).
• Smear technique: Smears can be made by ‘impression-smear’ method, spreading with platinum loop or crushing and spreading material with another side. Preparation can be fixed stained and examined (e.g. Examination of blood smears). They can be cleared and mounted in a mountant.
• Sectional methods: here anatomical relations are preserved and maintained and extremely thins sections are cut. Finished preparation is mounted in a medium with high refractive index to enable its detailed study. Medium that is used for embedding tissues depends upon technique employed and type of material to be sectioned. Most common medium is paraffin wax. In certain cases celloidin, and occasionally gelatin are used.
• Paraffin Technique: has the widest application. The tissue are embedded in a block of paraffin wax with melting point of 58^º to 60^ºC after subjecting them to various processes which render them permeable to molten paraffin wax.
• Frozen Sections: Unfixed or fixed tissue is frozen on a special microtome with carbon dioxide. This method is especially useful when rapid results are required or when demonstrating materials soluble in alcohol or cleaning agents like xylol.
• Vital staining: Some living cells take up certain stains (vital stains) which colour certain elements in cells (e.g. mitochondria). Some cells like phagocytes engulf microscopic coloured particles, which are then visible inside the cells (e.g. elements of R.E. system). Vital staining is subdivided into
• Supravital staining: This refers to staining living cells outside the body. Janus green and neutral red stains cytoplasmic organells like mitochondria. Living cells are treated with very dilute solutions of the stains. The cells are alive when they take up the stains but die subsequently as the stains are toxic.
• Intravital staining: Dyes like Trypan blue, Lithium carmine or Indian ink are injected subcutaneously or intravenously and tissues are collected from the site of injection (when subcutaneously injected) or pieces of organs containing phagocytic cells are prepared and processed by the usual methods. The dye will appear as fine granules in the cytoplasm of phagocytic cells.

Methods of examination of tissue

• Using optical microscope with direct or transmitted illumination.
• Using polarizing microscope.
• Using dark ground illumination.
• Using phase-contrast microscope to examine living cells and tissues without staining.
• Using electron microscope for obtaining magnification upto 1,00,000 times.
• Of these, the most common method is using ordinary optical microscope with direct or transmitted illumination.