196 LABORATORY EXERCISES IN BACTERIOLOGY 



degree than alcoholic solutions, which for the most part are employed only as counter- 

 stains or as stock solutions from which the watery solutions are prepared as required. 

 There is little difference manifested by different types of vegetating bacteria in the 

 assumption of the staining reagents, but some types require, in addition to the simple 

 solution of the dye, the previous or associated influence of substances like the alkalies, 

 aniline oil, acetic acid, or carbolic acid for the proper penetration of the coloring matter. 

 Such a substance is known as a mordant. Its precise mode of action is not clearly 

 understood and probably is not in all instances identical. It may involve the removal 

 of some oleaginous material from the bacterial capsule or the production of chemical 

 alterations in the composition of the latter, by which it becomes more pervious to the 

 staining fluid and thus allows the latter to enter the protoplasmic body of the bac- 

 terium. The organisms of tuberculosis and leprosy and the mycobacterium of smegma 

 are prominent examples which require the addition of such a mordant for effective 

 staining; and the spores of all bacteria show extreme refractiveness to the penetration 

 of stains, even in the presence of the common mordants. 



As a group bacteria are not so readily colored as are animal structures, and in 

 fact there is some difference between different varieties; and, as is the usual rule that 

 such substances as are with difficulty colored may be not readily decolorized, they are 

 apt to retain their coloration in the presence of decolorizing agents longer than asso- 

 ciated animal matter, which has been quickly and easily stained. So, too, some 

 difference, available in a few instances for diagnostic purposes, between different types 

 subjected to a definite decolorizer may be distinguished. Of these decolorizing agents 

 water is the type, but the least active ; its power of removal of stains is increased when 

 it is used hot. Alcohol is more energetic, a few minutes of exposure of stained films 

 being sufficient to discharge the tint from animal tissues as well as from many bacteria. 

 Among the more powerful decolorizing agents in common use are weak solutions of 

 acetic acid or of the mineral acids. 



The process of staining the films as above prepared is usually carried out by placing 

 a few drops -of the coloring solution on the film fixed upon the surface of the slide or 

 cover-glass (film side uppermost), contact being continued for from a half minute to 

 several hours in case of different organisms, varying with the type of organism, the 

 staining fluid used, and the application of heat (the process being much shortened when 

 the stain is applied hot). If one prefers, a cover preparation may be floated upon the 

 surface of the staining fluid, the film side downward, in contact with the stain. Every- 

 thing capable of being colored by the stain having been sufficiently tinged, the prepara- 

 tion is rapidly washed through water or one of the other decolorizing fluids so as to 

 discharge the color from everything in the film but the bacteria. Where several types 

 of organisms of different resistance to decolorizations exist in the preparation, the 

 differentiation is accomplished by permitting sufficient action of the decolorizer to 

 remove the stain from the less resistant, but not sufficient to accomplish the decoloriza- 

 tion of the more resistant. As a third step of the process, one may, for greater ease of 

 detection of the stained organisms in the preparation, quickly apply a contrast stain to 

 those elements from which the decolorizing fluid has discharged the first stain. Eosin, 

 vesuvin, methylene-blue, safranin, and Bismarck brown in alcoholic solution are usually 

 chosen for this purpose. 



These aniline stains are not so permanent as hematoxylin or carmine stains; 

 preparations are best preserved if kept in closed boxes away from the light. 



