LEAFLET IV 



STAINING METHODS 



9th Edition 



General Principles 



The staining of bacteria depends in general upon the same prop- 

 erties of dyes as docs the staining of animal or plant tissue for histo- 

 logical purposes. Short discussions of the nature of dyes, with special 

 reference to staining are given elsewhere (Conn, 1940; Churchman, 

 1928) 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 ow^e their colored properties 

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

 aqueous solution 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. As bacteria do 

 not show typical cell structure and the nuclear material seems to be 

 distributed throughout their bodies, they tend to stain fairly uniform- 

 ly with nuclear, i. e., the basic, dyes. Hence, the stains in common 

 use by the bacteriologists are rarely acid dyes. 



PREPARATION OF SMEARS 



Pure cultures of bacteria can ordinarily be prepared for staining 

 by the simple process of making an aqueous suspension and drying 

 a drop of it on a slide or cover glass, without any fixation other than 

 gentle heat. The use of this simple procedure depends upon the fact 

 that most bacteria, because of their small size or their stiff walls, 

 can be dried without great distortion. For this reason it is not 



