MICROSCOPICAL EXAMINATION OF BACTERIA 99 



The Principles involved in Staining. The development of 

 the technique of staining has been largely empirical, and at the 

 present time only the most general of the principles involved are 

 understood. In fact, it is not known whether the theory of 

 staining rests upon a chemical or a physical basis. There is 

 some evidence, as will be seen, to show that the principle 

 involved in staining is merely a physical one. There is other 

 evidence that, at least in certain cases, the reaction between 

 the dye and protoplasm may be a chemical one. In a general 

 way it seems true that the staining of the protoplasm is re- 

 lated to the question of diffusion, and those conditions which 

 aid diffusion aid also in staining. In ordinary routine work, 

 the dried bacterial cells are stained with an aqueous solution of 

 the dye, and concentrated alcoholic solutions are not satisfac- 

 tory for staining dried bacteria. There is diffusion between the 

 dried bacterial cell and the watery solution, and none in the 

 case of dried bacteria and alcohol. If the bacteria are first 

 wet, then they will stain with the alcoholic solutions. The 

 majority of bacteria stain readily with the ordinary aqueous 

 solution of the dyes. To stain certain of them, however, it is 

 necessary to resort to special means. Some of the bacteria, 

 as we have seen, have certain fatty or waxy substances in their 

 cell walls, which render their staining by watery solution of the 

 dyes difficult or well-nigh impossible. When they are once 

 stained, however, it is difficult to destain them. For this 

 purpose even the mineral acids may be used. These bacilli are 

 known as the acid-fast bacilli. In order to stain them it is 



