Appendix IV: Comments on Color Groups, Stains, and the Staining Mechanism

Ronald A. Bergman, Ph.D., Adel K. Afifi, M.D., Paul M. Heidger, Jr., Ph.D.
Peer Review Status: Externally Peer Reviewed

The chemical mechanism by which biological stains impart color to specific cellular components and tissues is poorly understood in many instances, although it has been the subject of numerous publications and books. The student whose interest or curiosity lies in this area is directed to the references.

Students frequently hear or read that certain dyes are either basic or acidic and that certain structures are basophilic or acidophilic. A detailed account of these and other terms can be found in H. J. Conn's Biological Stains, by R. D. Lillie, cited in the references. A very brief explanation seems appropriate here, and a discussion of both a synthetic and a natural dye follow.

Known atomic groupings in molecules related to color are called chromophores. These atomic groupings include the azo group (N==N), the azin group and the inclamine group (N==), and are known as the basic chromophores. The nitro group (NO²), and the quinoid benzene ring with its CXC groups constitute the important acidic chromophores. Other chromophores are not usually of biological interest. All of the so-called coal tar or synthetic dyes are derivatives of benzene, an important organic compound capable of combining in an infinite number of ways with radicals and elements to form complex compounds. Benzene compounds with which chromophores are combined are known as chromogens. Chromogens, although colored, are not dyes until they possess other end groups that permit them to form a chemical union with tissue acidic or basic end groups. Synthetic dyes are prepared so that the essential part of the dye is either acid (anionic) or basic (cationic) in its chemical behavior. Dye solutions are prepared from dye salts, which, when placed in water, yield by dissociation either hydroxyl (OH^-) ions and act as cations (basic dyes) or hydrogen (H⁺) ions and act as anions (acidic dyes). A cationic or basic dye is a salt (usually chloride) of a chromogen base and will stain the nucleic acids of the nucleus and cytoplasm, whereas an anionic or acid dye is a salt (usually sodium) of a chromogen acid and will stain the cytoplasm of cells (except cytoplasmic ribonucleoprotein) and certain other tissues. A basic dye has an affinity for nuclei, which are then termed basophilic structures, and an acid dye has an affinity for non-nucleic acid-containing cytoplasm and other tissues, which are then termed acidophilic structures.

Eosin Y is an anionic or acid (synthetic) dye in spite of the fact that a solution of the sodium salt of eosin is basic in reaction. It is important to recognize that eosin is an acid or anionic dye, because the significant part or chromogen of the dye that forms the union with cellular or tissue cationic or basic end groups is anionic or acidic. When eosin is used as a tissue stain, the acidophilic structures are sometimes referred to as eosinophilic.

Hematoxylin is a natural dye extracted from the logwood tree, Hernatoxylin campechium, found in Central and South America. The extract from the heartwood is not a dye until it undergoes oxidation into hematein, which is an acid chromogen. In this form, hematein is only a weak dye with little affinity or specificity. Mordanting is essential to the conversion of this dye to a base, which will then combine actively and strongly with acidic nucleic acids. Mordants most commonly used in tissue staining are salts of aluminum, chromium, iron, potassium, and tungsten.

Another type of staining reaction is metachroming, in which a dye, such as toluidine blue, will react with certain components of cells and tissues and have a different hue from its original blue color. Metachromatic staining is seen in mast cell granules, cartilage, and goblet cell mucus, which will appear red-purple when stained with toluidine blue. The nuclei of these cells appear blue, the same color as the original dye solution. The principal reason for metachromatic staining is found in the chemical nature of the components of these cells and tissues, which contain a predominance of strongly acidic sulfated protein polysaccharides.

Additional comments about stains and staining reactions are given in the legends of some of the illustrations in this atlas.

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