184 BACTERIOLOGY 



To 100 c.c. of a saturated alcoholic solution of methylene 

 blue add 1 gram of rosolic acid and 20 c.c. of glycerine. 

 The bacilli are stained red, the balance of the field blue. 



Gram's Method. Another important differential method 

 of staining which is very commonly employed is that recom- 

 mended by Gram. In this method the objects are treated 

 with an aniline-water solution of gentian-violet made after 

 the formula of Koch-Ehrlich. After remaining in this for 

 two or three minutes they are immersed in a solution com- 

 posed of 



Iodine 1 gram 



Potassium iodide . . . . . . . . . 2 grams 



Distilled water . . . . . ..... . 300 c.c. 



In this they remain for about five minutes; they are then 

 transferred to 95 per cent, alcohol and thoroughly rinsed. 



This method is particularly useful in demonstrating the 

 capsule which is seen to surround some bacteria, especially 

 micrococcus lanceolatus of pneumonia. 



After such treatment certain species of bacteria are found 

 to be of a very dark purple color, while all else in the prepa- 

 ration is decolorized; other species lose their color entirely 

 in the process. Those that retain the dark stain are com- 

 monly denominated as " Gram-positive" while those that 

 lose their color are known as "Gram-negative." While the 

 majority of bacteria are either definitely positive or negative 

 to this reaction, there are a few species that are indeter- 

 minate in this particular, that is to say, they become partly 

 decolorized and one cannot say certainly that they are 

 either positive or negative. Under certain conditions of 

 cultivation, and especially under conditions favorable to 

 degenerative changes, some species that are normally 



