METHODS OF OBSERVING BACTERIA. 155 



decolorant and must be washed out in xylol before the 

 section is mounted in Canada balsam. 



Gram's method is chiefly employed for staining bac- 

 teria in tissues, but the fact that not all bacteria are 

 colored by it is of considerable differential import- 

 ance, as the difficulty of separating the species of 

 bacteria is so great that use must be made of every 

 aid. 



Gram's method for cover-glass preparations is 

 employed for differentiating between different species 

 of bacteria. A thin layer of the bacteria to be exam- 

 ined is spread upon the cover-glass, dried, and fixed. 

 The cover, held in the grip of a cover-glass forceps, is 

 flooded with Ehrlich's solution. The solution is kept 

 warm by holding the cover flooded with the stain over 

 a small flame. The process of staining is continued from 

 two to five minutes. If the heating causes the stain to 

 evaporate, more of it must be dropped upon the glass, 

 so that it does not dry up and incrust. 



The stain is poured off, and the cover placed in a small 

 dish of Gram's solution and allowed to remain one-half 

 to two minutes, the solution being agitated. It is pos- 

 sible to apply the Gram solution in the same manner 

 in which the stain is used, but as a relatively larger 

 quantity should be employed, the dish seems preferable. 



The cover is next washed in 95 per cent, alcohol until 

 the blue color is wholly or almost lost, after which it can 

 be counter-stained with eosin, Bismarck brown, vesuvin, 

 etc., washed, dried, and mounted in Canada balsam. 

 Given briefly, the method is : 



Stain with Ehrlich's solution two to five minutes ; 

 Gram's solution for one-half to two minutes ; 

 Wash in 95 per cent, alcohol until decolorized ; 

 Counter-stain if desired ; wash the counter-stain 



off with water ; 

 Dry; 

 Mount in Canada balsam. 



