MICROSCOPIC METHODS 71 



for these dyes and to note their importance as a means of differentiating 

 micro-organisins from other corpuscular elements. 



The staining of bacteria is not to be considered simply as a mechani- 

 cal saturation of the cell body with the dye, in which the latter is dis- 

 solved in the plasma. It is rather a chemical combination between the 

 dye substance and the plasma. This union, however, is apparently 

 an unstable one and easily broken up. Unna believes that the basic 

 aniline dyes, from their chemical composition, are not really bases but 

 neutral salts e.g., fuchsin equals rosaniline chloride; they are called 

 basic only because the staining components (as the rosaniline) are of a 

 basic nature. The staining process is, therefore, not to be looked upon 

 as if the dye substance separated into its component parts and only the 

 staining ingredient attacked the cell body, because the tissues for which 

 these "basic aniline dyes" have special affinity are themselves basic. 

 On the contrary, the dyestuff unites as a whole with the plasma, form- 

 ing, as it were, a double salt or unstable compound between the two. 



The dependence of the staining process upon the solvent condition 

 of the dye is shown in the following observations : 



1. Entirely water free, pure alcoholic dye solutions do not stain. 



2. Absolute alcohol does not decolorize bacteria, while diluted alcohol 

 is an active decolorizing agent. The compound of dye substance and 

 plasma is therefore insoluble in pure alcohol. 



3. The more completely a dye is dissolved the weaker is its staining 

 power. For this reason pure alcoholic solutions are inactive; and the 

 so-called weak dye solutions to which strong dye solvents have been 

 added are limited in their action on certain bacteria in which the dye 

 substance is closely united. This is the principle of Neisser's stain for 

 diphtheria bacilli viz., acetic acid methylene-blue solution. 



On the other hand, the addition of alkalies to the dye mixture ren- 

 ders the solvent action less complete, leading to slight clouding, and 

 finally, if large amounts of alkali are added, to precipitation of the 

 dye substance. Solutions thus treated possess an intense staining 

 power. According to Michaels, however, in Loeffler's methylene- 

 blue solution the role of the alkali is purely of a chemical nature, by 

 which it converts the methylene blue into methylene azure (azure II). 



The dependence of the staining process upon the nature of the bac- 

 teria is exhibited in the following facts: 



There are among bacteria some which are easily stained and others 

 which are only stained with difficulty. To the latter belong, for ex- 

 ample, the tubercle bacillus and lepra bacillus, also spores and flagella. 

 The difference between them is that the easily stained objects require 

 but a minimum of time to be immersed in a watery solution, while 

 the others must be stained by special dyes or with the aid of outside 

 influences (heat and mordants, etc.). The difficultly stained objects 

 are at the same time not easily decolorized. The explanation of the 

 resistance which these bacteria show to staining as well as to decolorizing 

 agents is to be sought in two ways: either on the assumption that they 

 possess a difficultly permeable or resisting envelope, or that they have 



