110 Stains and Staining (chap. 10) 



and triple staining, the use of two or more stains on the same slide. By 

 placing several different contrasting colors in a tissue, this type of stain- 

 ing has definite advantages. The dyes, if properly chosen, will stain 

 histologically; that is, each dye because of a known specificity will elect 

 to stain only specific parts of the cells. Due to their chemical nature 

 synthetic dyes make this kind of staining possible. By being synthesized, 

 their formula can be controlled and the significant part of the dye is 

 either anionic (acid) or cationic (basic) in action. Actually dye powder 

 as purchased is a salt, biu the salts of the so-called basic dyes give up 

 OH~ ions and act as cations, while the acidic dyes give up H+ ions and 

 act as anions. Therefore an acid dye is the salt of a color acid, usually a 

 sodium salt; a basic dye is the salt of a color base, usually a chloride. 

 Basic dyes have an affinity for nuclei, which are basophilic (readily 

 stained by basic dyes) and acidic dyes have an affinity for the cytoplasm, 

 which is acidophilic (readily stained by acidic dyes). Also acid and base 

 dyes may be combined to form "neutral dyes" which give results differ- 

 ing from those obtained with ordinary double staining using separate 

 acid and base dyes. The action of neutral dyes is an example of poly- 

 chroming, a process in which a dye forms other dyes spontaneously — 

 the basis of the development of modern blood staining (Romanowsky 

 stains) in which methylene blue and eosin combine in a polychroming 

 mixture. Through polychroming, a new group of dyes, azures, is pro- 

 duced for the multiple-staining action which is so desirable in the differ- 

 entiation of white blood cells. 



Polychroming must not be confused with another type of staining, 

 metachroming, in which certain substances are stained in one color 

 and others in another color by the same dye. In the case of the dye 

 thionin, the explanation for this reaction may be as follows: 



Thionin stains chromatin blue; it stains mucus, ground substance of 

 cartilage, and granules of mast cells, red. The dye seems to exist in aqueous 

 solution in two forms: (I) the normal color, blue; and (2) the metachromatic 

 color, red. Both forms are always present, but the red is in a polymerized 

 form of the blue. The blue form is favored by increase of temperature, a 

 lowering of pW, a decrease of dye concentration, and addition of salts, al- 

 cohol, or acetone. The red form is favored by a decrease of temperature, a 

 raising of pH or increase in concentration of dye {Bergeron and Singer, 

 1958). Also certain substances, sulfuric esters of high molecular weight and 

 their salts, increase the production of the red form. Mucin, the ground sub- 

 stance of cartilage, and the granules of mast cells contain substances of this 

 nature, and therefore take up the metachromatic form. 



