LESSON v BACILLUS ANTHBACIS 23 



(5) Inoculate a broth tube and place it in a water 

 incubator at 42 -5 C. 



Examine it microscopically a week later : no spores 

 will be found, and the bacillus, moreover, is 

 attenuated and less virulent. 



(6) Inoculate two neutral litmus agar-agar tubes : one 

 with ordinary virulent bacilli and the other with the 

 attenuated bacillus. 



The latter produces less acid. 



(7) (a) Inoculate a tube containing a little sterilised *75 

 per cent saline solution with three platinum loops of an 

 agar-agar culture of Bacillus anthracis. 



(b) Shake the inoculated tube vigorously, and from it, 

 with a platinum loop, inoculate a sloped gelatine tube, 

 thoroughly streaking the surface of the culture medium. 



(c) Place the tube in the cool incubator, and when small 

 colonies appear they are to be worked up by means of 

 impression specimens (vide infra p. 26). 



(8) Staining of Bacillus anthracis. 



(a) Prepare cover-glass specimens in the usual manner 



(1) of a broth culture of Bacillus anthracis; 



(2) of a gelatine culture ; 



(3) of an agar-agar culture. 



(b) Stain films 



(a) With Loffler's methylene-biue after having cleared 

 with acetic acid. 



(/3) With gentian-violet after having cleared with warm 

 water. 



