EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 4! 



The watery solutions of aniline dyes prepared as above de- 

 scribed deteriorate in a short time, and it is best to prepare 

 them freshly each time they are required. A very useful 

 solution, which is permanent, is Loffler's alkaline methylene- 

 blue: 



Concentrated alcoholic solution of methylene-blue. . 3Oc.c. 

 Potassium hydrate (caustic potash) 1-10,000 

 watery solution 100 c.c. 



Loffler's methylene-blue is a good stain for general pur- 

 poses. It is perhaps more in use than any other formula 

 for coloring the diphtheria bacillus. 



Aniline-water Staining Solutions. The intensity with 

 which aniline dyes operate may be increased by adding 

 aniline oil to the solution : 



Aniline oil 5 c.c. 



Water 100 c.c. 



Mix, shake vigorously, filter; the fluid after filtration 

 should be perfectly clear; add 



Alcohol 10 c.c. 



Alcoholic solution of fuchsin (or gentian-violet, or 

 methylene-blue) 1 1 c.c. 



Aniline-water staining solutions do not keep well, and 

 need to be freshly prepared about every two weeks. The 

 applications of the aniline-water stains will be given under 

 separate headings. In general, however, they are em- 

 ployed where a stain of unusual power is required. 



Gram's Method. Cover-glass preparations, having been 

 prepared and fixed in the usual manner (see page 37), are 

 stained as follows : 



(a) Stain in aniline-water gentian-violet solution, from 

 two to five minutes. The intensity of the stain may be in- 

 creased by warming slightly. 



(b) Iodine solution, one and one-half minutes : 



