2.5. Staining

Most biological tissues have very little contrast, and cellular details are hard to discern with the ordinary light microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. Plant histologists have been staining tissues with natural dyes but today most dyes are synthetic. Dyes are extensively used in histology as they can enhance and improve the visibility of the specimen and often have an affinity for a specific tissue element, allowing quick and easy identification of specific cell types and cellular components. Although some dyes can be used on their own to stain tissues, specific dyes are often used with a counter stain to contrast various components within the tissues. Prior to staining, it is necessary to de-wax sections; resin sections can be stained directly.

Tolonium chloride (also known as toluidine blue) is a cationic dye that binds to negatively charged groups. An aqueous solution of this dye is blue, but different colors are generated when the dye binds with different anionic groups in the cell. For example, a pinkish purple color will appear when the dye reacts with carboxylated polysaccharides, such as pectic acid; green, greenish blue or bright blue with polyphenolic substances, such as lignin and tannins; and purplish or greenish blue with nucleic acids. Acridine orange is a nucleic acid selective fluorescent cationic dye that interacts with DNA and RNA by intercalation and electrostatic attractions, respectively. It is useful as a non-specific stain for backlighting conventionally stained cells. However, extreme care should be taken when using Acridine orange as it is carcinogenic. Haematoxylin is extracted from the heartwood of the logwood tree. It is one of the most commonly used stains in histology. It is generally utilized with a second counter stain, where haematoxylin as the primary stain for nuclei and contrasting or counterstaining with Orange G, safranin, or Fast Green.

 

2.5.i. Cell wall stains: Although cell walls can be visualized using general tissue stains such as Toluidine blue, there are a number of quick and simple methods which can be used to specifically stain individual cell wall components, such as pectin using Ruthenium red, cellulose using Calcofluor, lignin using Phloroglucinol or safranin and callose using Aniline blue, Resorcinol blue or Astra-blue.