LABORATORY WORK WITH ANTHRAX ANIMALS. 291 



bacillus grown in stab culture in gelatin, in bouillon, on 

 ordinary agar, and on peptonless agar. The preparation 

 of impression cover-glasses of the anthrax colonies and 

 simple stains of the bouillon hanging-drop culture have been 

 mentioned. 



Double staining of spores. The growth of the anthrax 

 bacillus on peptonless agar, or on potato at 37, when exam- 

 ined in the hanging-drop, will show the presence of an 

 abundance of bright, highly refracting oval bodies or 

 spores. These may be observed free and also within the 

 parent cell. Simple stains of this material with fuchsin, 

 etc., will show the bacilli deeply stained, whereas the 

 spores remain colorless. This is undoubtedly due to a 

 special composition of the spore contents, as well as to the 

 dense impenetrable wall which surrounds the spore and 

 prevents the dye from passing into the interior (p. 55). By 

 proper treatment with strong anilin dyes it is possible to 

 force the stain into the spore. Once within the spore, it is 

 as -difficult to remove the dye as it was to cause it to enter. 

 By suitable decoloration it is, therefore, possible to remove 

 the stain from everything on the cover-glass, except from 

 the spores. Then, on the application of a contrast color 

 the specimen will show a bright red spore within a blue bac- 

 illus. The method of double staining spores is as follows: 



The cover-glass preparation from the peptonless agar 

 is dried in the air and fixed in the usual manner. The 

 cover-glass is held in the forceps in the left hand, with the 

 specimen side up, and covered with a solution of hot car- 

 bolic fuchsin. This is held over a Bunsen flame, so that 

 vapors are given off from the liquid. Active ebullition 

 should be avoided. - From time to time the liquid which is 

 lost by evaporation is replaced by a fresh addition of the 

 carbolic-fuchsin, and under no condition should the dye be 

 allowed to dry down on the cover-glass. The best results in 

 heating are obtained when the flame is turned low, so that 



