CHAPTER II 

 Staining Methods 



H. J. Conn in collaboration with J. W. Bartholomew and 

 M. W. Jennison 



GENERAL PRINCIPLES 



The staining of bacteria depends in general upon the same properties 

 of dyes as does the staining of animal or plant tissue for histological pur- 

 poses. Short discussions of the nature of dyes, with special reference to 

 staining are given elsewhere (Conn, 1953), and only the briefest summary 

 of the subject need be given here. 



All bacterial dyes are synthetic products — anilin dyes, or coal-tar dyes, 

 as they are generally called. Although the synthetic dyes vary greatly 

 in their chemical nature and staining properties, they are for practical 

 purposes often divided into two general groups, the acid dyes and the 

 basic dyes. These terms do not mean that the dyes in question are free 

 acids or free bases. The free color acids and bases, when obtainable, are 

 colored, to be sure, but they are often insoluble in water and rarely have 

 appreciable staining action; i.e., the colors do not ''stick." The salts of 

 these compounds, on the other hand, are more soluble, penetrate better, 

 and stain more permanently; they are the true dyes. 



An acid dye is the salt of a color acid; a basic dye the salt of a color 

 base. In other words, acid dyes owe their colored properties to the 

 anion, basic dyes to the cation. The actual reaction of an aqueous solu- 

 tion of a dye, however, depends on several factors, and an acid dye may 

 well be basic in reaction, while a basic dye may be acid. This is because 

 the reaction of such a solution depends on the relative strengths of the 

 dye ion and of the anion or cation with which it is combined in the dye 

 salt. 



Basic dyes have greatest affinity for the nuclei of cells, probably because 

 of the acid nature of the nuclear material. Acid dyes have a stronger 

 tendency to combine with the cytoplasm. Bacteria do not show typical 

 cell structure, and they tend to stain fairly uniformly with nuclear, i.e., 



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