256 BACILLUS OF SYMPTOMATIC ANTHRAX 



form then appears blue, the clostridia black violet, and the spores 

 remained unstained. In the bodies of sick cattle spore formation is 

 generally well marked; the contrary is true in guinea-pigs infected 

 experimentally. The bacillus keeps the stain when treated by Gram's 

 method. 



Cultural Properties. The black-leg bacillus is a strictly anaerobic 

 bacterium. According to Kitt the best method of obtaining pure 

 cultures is the following: With a very fine funnel, gelatin and agar 

 are poured into glass tubes 20 to 30 cm. long and 3 to 5 mm. wide, 

 which furnishes a high narrow column of the medium. These glass 

 tubes are fused at one end and closed at the other with a cotton plug. 

 From four to ten of these agar and gelatin tubes are melted and cooled 

 in the usual manner and then inoculated; the first one with a few 

 drops of meat juice or blood from a case of black-leg, the others in 

 the ordinary diluting manner, i. e., No. 2 is inoculated from No. 1, 

 No. 3 from No. 2, and so forth. In the last tube, No. 10, for example, 

 there is so little of the original bacilli-containing material that a slow 

 growth, with scanty colony formation, will occur. Whenever more 

 abundant material is used in inoculation the growth in the incubator 

 under anaerobic conditions is so rapid that no individual colonies 

 are formed and the culture medium is broken up as a result of the 

 extensive gas formation. In successful attempts with very high 

 dilutions delicate, grayish, very finely granular, or punctate colonies 

 from 0.5 to 1 mm. in diameter develop. They sometimes become 

 larger, more compact, and less transparent, and attain the size of 

 a millet seed. The growth never reaches the surface of the medium. 

 In gelatin the colonies somewhat resemble frog's spawn; they liquefy 

 the medium completely, except the uppermost zone, which is not 

 invaded and remains solid. In the liquefied mass the growth sinks 

 to the bottom and a whitish, rather scanty cloudy sediment collects. 

 The addition of cattle serum to the gelatin is very favorable to the 

 growth. In nutrient bouillon containing some sterile cattle blood 

 serum the growth is abundant, the medium soon becomes cloudy, 

 and gas is formed. A medium composed of bouillon (10 c.c.), to 

 which 1 to 5 c.c. of fresh sterile blood obtained from the live animal 

 has been added, enables the bacillus to grow in the presence of air. 

 A good growth can also be obtained by placing a piece of fresh 

 muscle (thorax muscle of a pigeon) procured in a sterile manner in 

 the inoculated bouillon. The bacillus also grows in pure sterile 

 blood. In this medium it forms spores abundantly and retains its 

 virulency much better than in other media, in which it generally 

 soon loses its pathogenic properties. 



Animals Susceptible. The bacillus is pathogenic to guinea-pigs, 

 very slightly to rabbits, which, on the contrary, are very susceptible 

 to the bacillus of malignant edema. In natural infection of cattle 

 the bacilli gain entrance through wounds of the skin into which mud 

 from marshy grounds has penetrated. This was demonstrated long 



