METHODS OF EXAMINATION 349 



pustule, and stained with a watery solution of methylene-blue 

 and also by Gram's method. By this method practically con- 

 clusive evidence may be obtained; but sometimes the result 

 is doubtful, as the bacilli may be very few in number. 

 McFadyean's methylene-blue method (p. 333) should also be 

 applied. In all cases confirmatory evidence should be obtained 

 by culture. Occasionally bacilli are so scanty that both film 

 preparations made from different parts and even cultures may 

 give negative results, and yet a few bacilli may be found when 

 a section of the pustule is examined. It should be noted that 

 the greatest care ought to be taken in manipulating a pustule 

 before excision, as the diffusion of the bacilli into the 

 surrounding tissues may be aided and the condition greatly 

 aggravated. The examination 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. 



(h] 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 recognised from their 

 wavy margins by means of a hand lens. They should also be 

 examined microscopically by means of film preparations. 



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

 should be mixed with some sterile bouillon or water, and 

 injected subcutaneously into a guinea-pig or mouse. If anthrax 

 bacilli are present, the animal usually dies within two days, with 

 the changes in internal organs already described. The diagnosis 

 of an organism as the anthrax bacillus cannot be said to be 

 substantiated till its pathogenicity has been proved. 



