SBPMD Histology Laboratory ManualHistological TechniquesIn order to study tissues with a microscope they must be preserved (fixed) and cut into sections thin enough to be translucent. The process of fixation is briefly described in the next section. Fundamentally it consists of a chemical or physical method of killing the tissue and yet retaining characteristic peculiarities of shape and structure. Following fixation, blocks of tissue must be cut into thin sections. One way is to make a firm block by freezing fresh or fixed tissue. Other techniques involve dehydration in alcohols and infiltration with paraffin, or some similar agent - a process called embedding. Sections 3 to 10 microns (3 to 10 thousandths of a millimeter) in thickness are cut on steel knives mounted in an instrument called a microtome, which has a precise mechanical advance. For electron microscopy the sections are considerably less than one ten-thousandth of a millimeter (0.1 micron, µm) thick. This is accomplished by embedding the tissue in a plastic such as Epon or araldite (epoxy resins) and cutting on special ultramicrotomes equipped with a fine mechanical or thermal advance. Sections are cut with glass or diamond knives and mounted on copper mesh grids. In work with the light microscope, it is difficult to recognize the various components of cells and tissues without differential staining. The stains may react chemically or physically and a wide variation is possible. The staining method can be altered to suit the needs of the examiner in order to accentuate certain tissues or organelles. Finally, in order to preserve the section which has been made from a block of fixed tissues and stained, it is mounted on a glass slide and covered with a thin cover glass by means of a transparent substance which hardens and seals the preparation to make it permanent. Some tissues are stained and then mounted. More often the tissue is placed on the slide first and then stained. The mounting medium used to attach the coverslip must have a refractive index similar to that of the glass slide and cover slip to prevent distortion. Slide Preparation Techniques |