EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 31 



headings. In general, however, they are employed where a 

 stain of unusual power is required. 



Gram's Method. The advantages .of Gram's method are 

 that by using it certain kinds of bacteria may be stained a violet 

 color, while other bacteria are stained feebly or not at all. 

 Cover-glass preparations, having been prepared preferably 

 from agar slant cultures twelve to twenty-four hours old* 

 and fixed in the usual manner (see pages 26 and 27), are 

 stained as follows. Some advise short rinsing with water 

 after pouring off the staining solution. 



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

 to five minutes. The intensity of the stain may be increased 

 by warming slightly. 



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



Iodine i gram. 



Potassium iodide 2 grams. 



Water 300 c.c. 



In this solution the preparation becomes nearly black. 



(c) Wash repeatedly in strong, preferably in absolute 

 alcohol; the alcohol becomes stained with clouds of violet 

 coloring matter; the alcohol is used as long as the violet color 

 continues to come away, and until the preparation is decolor- 

 ized or has only a faint gray tint. It may be necessary to repeat 

 the treatment with the iodine solution. 



(d) When desired, the specimens may be stained, by way of 

 contrast, with a watery solution of Bismarck brown or eosin. 



(e) Wash in water, and examine either in water ^directly or 

 after drying and mounting in Canada balsam. Gram's 

 method and its modifications are not always trustworthy for 

 diagnostic purposes; since one and the same organism may 

 stain but faintly if all at one time, and quite intensely at 

 others.! Still it is a more or less useful aid, particularly with 



*Muir and Ritchie. American Ed., 1903, p. 103. Also Mallory and Wright. 

 1904, p. 101. 



fKolle and Wassermann. Vol. I, p. 70. 



