METHODS OF EXAMINATION. 295 



malignant pustule, film preparations should be made from 

 the fluid in the vesicles or from a scraping of the incised 

 or excised pustule, and stained with a watery solution of 

 methylene-blue and also by Gram's method. By this 

 method practically conclusive evidence may be obtained ; 

 but sometimes the result is doubtful, as the bacilli may be 

 few in number. In all cases confirmatory evidence should 

 be obtained by culture. It should be noted that the greatest 

 care ought to be taken in handling the part, as otherwise the 

 diffusion of the bacilli into the surrounding tissues may be 

 aided and the condition greatly aggravated. The examina- 

 tion of the blood in cases of anthrax in man usually gives 

 negative results, with the exception of very severe cases, 

 when a few bacilli may be found in the blood shortly before 

 death, though even then they may be absent. 



(b) Cultivation. A small quantity of the material used 

 for microscopic examination should be taken on a platinum 

 needle, and successive strokes made on agar tubes, .which 

 are then incubated at 37 C. At the end of twenty-four 

 hours anthrax colonies will appear, and can be readily recog- 

 nised from their wavy margins, by means of a hand lens. 

 They should also be examined microscopically by means of 

 film preparations. 



(c) Test Inoculations. A little of the suspected material 

 should be mixed with some sterile bouillon or water, and 

 injected subcutaneously into a guinea-pig, or it may be intro- 

 duced into the subcutaneous tissue by means of a seton. If 

 anthrax bacilli are present, the animal usually dies within 

 two days, with the changes in internal organs already 

 described. 



