LABORATORY WORK WITH ANTHRAX ANIMALS. 287 



Stain some of the fixed cover-glasses with simple anilin 

 dyes, such as gentian violet or fuchsin, observing the sug- 

 gestions made on p. 281. Examine and study the specimen^ 

 carefully and make permanent preparations. Note the or- 

 gan in which the bacilli are most abundant. The remain- 

 der of the cover-glasses will serve for double-staining by 

 Gram's method. 



Stained preparations of the heart-blood (p. 280), should 

 also be made. 



Gram's method. This excellent method of demonstrating 

 the presence of certain bacteria in the fluids and tissues of 

 the body is based upon the fact that the protoplasm of the 

 bacterial cell, when stained with anilin-water gentian violet 

 and then treated with iodine, forms a difficultly soluble com- 

 pound. By proper exposure to a solvent the dye can now 

 be removed from the entire cover-glass, but not from the 

 bacterial cell. The deeply stained violet bacteria lie on a 

 colorless back-ground which on treatment with a contrast 

 color, as eosin or picro-carmin, becomes stained a light 

 pink. The method is as follows: 



A solution of anilin-iuater gentian violet is first prepared. 

 Anilin oil is placed in a test-tube to a depth of about half 

 an inch. The tube is then filled with water, closed with the 

 thumb, and thoroughly shaken in order to obtain a satur- 

 ated aqueous solution of anilin. The liquid is then passed 

 through a small filter and collected in another test-tube. 

 The filtrate should be perfectly clear, not cloudy. To the 

 anilin water thus obtained a saturated alcoholic solution of 

 gentian violet is added till the fluid is deeply colored, and 

 opaque. This result is obtained when 0.3 0.5 c.c. of the 

 saturated gentian violet is added to 10 c.c. of the anilin- 

 water. 



Some of the anilin-water gentian violet thus prepared 

 is poured out into a watch-glass, or better, into a wide Es- 

 march dish. The fixed cover-glass is placed between the 



