MORPHOLOGY 555 



swellings, by smearing a little of the peritoneal exudate or oedema fluid on a 

 cover-glass, or by rubbing a cover-glass lightly over the surface of the liver 

 of a guinea-pig dead of the disease. These films may be stained with carbol- 

 crystal-violet, Gram's stain or Claudius' stain. 



(c) Sections. The swellings should be used for cutting sections and are 

 best stained with Kuhne's carbol-blue or by Gram's method with counterstain. 



Note. The tissues of carcases dead of symptomatic anthrax rapidly become 

 invaded by the bacillus of malignant oedema, a normal inhabitant of the intestine. 



2. Cultural characteristics. 



Conditions of growth. The bacillus of quarter ill is a strict anaerobe 

 and must therefore be cultivated by the methods applicable to anaerobic 

 organisms. 



The media should be recently prepared. Ordinary broth is not a good 

 medium and though the addition of glycerin (4 per cent.), glucose (1 per 

 cent.), or sulphate of iron (1 per cent.), etc. has been proposed, such additions 

 are said to offer no advantages (Kitasato). Leclainche and Vallee obtained 

 very good results with recently prepared Martin's broth. A considerable 

 amount of gas is formed in culture (H, C0 2 , CH 4 ) which has a characteristic 

 odour of butyric acid. 



Growth commences at 15 C. the optimum temperature being about 37 C. 



For isolating the organism the peritoneal exudate or heart blood or some of the 

 fluid from the swelling should be used for sowing cultures, but it is rather difficult 

 to obtain pure cultures : in the pulp from the swellings the bacillus is almost 

 always associated with other organisms (facultative aerobes, Vibrion septique, etc,). 



It is necessary also to test the purity of the culture from time to time by micro- 

 scopical examination (as indicated above, the appearance of long and wavy threads 

 denotes a contamination with the Vibrion septique) and by the inoculation of a 

 rabbit and guinea-pig. If only the guinea-pig die the virus is pure and four or five 

 drops of blood from the heart should be sown immediately after death this affords 

 in any case a certain method of obtaining pure cultures. 



Maintenance of virulence. In the laboratory the virulence of the micro-organism 

 is maintained by passage through guinea-pigs. It should be remembered that 

 infection with the Bacillus chauvcei predisposes in a marked degree to infection 

 with the Vibrion septique and that after a few passages the animals die of a double 

 infection. 



Culture media. Broth. Recently prepared Martin's broth is better than 

 ordinary broth. After incubating for about 20 

 hours at 37 C. there is a general cloudiness of 

 the medium and gas is given off in considerable 

 amount. After 2 or 3 days flocculi form which fall 

 to the bottom and the broth gradually becomes 

 clear. 



Albuminous media. The growth is more luxur- 

 iant and the virulence is maintained for a longer 

 time than in broth. Serum or serum diluted 

 with two parts of sterile water or meat juice 

 prepared according to the method given when 

 dealing with the Vibrion septique should be utilized. 



Gelatin. Deep stab cultures. Small irregularly FIG. 263. The bacillus of quar- 

 spherical colonies throwing out lateral offshoots ^ t k I ffi e ^ OTa * UC( ^ 

 slowly make their appearance along the line of the 



needle puncture when the medium is incubated at 20 C. : when they begin 

 to become confluent the gelatin is split by the formation of gas bubbles. The 

 culture spreads irregularly and liquefies the medium. 



