ISO LA TION OF TE TANUS BA CILL US. 355 



free in the culture medium. They can be stained by the 

 appropriate methods. 



Isolation. The isolation of the tetanus bacillus is some- 

 what difficult. By inoculation experiments in animals, its 

 natural habitat has been proved to be garden soil, and especi- 

 ally the contents of dung -heaps. From such sources, and 

 from the pus of wounds in tetanus, it has been isolated by 

 means of the methods appropriate for anaerobic bacteria. 

 It probably leads a saprophytic existence, but its function 

 as a saprophyte is unknown. Often, however, instead of 

 soil, etc., being taken as the original material, there is 

 employed the pus locally developed in animals dead of the 

 disease after inoculation with garden earth, etc. The best 

 methods for dealing with such pus or with that derived from 

 wounds in the natural disease are as follows : 



(i) The principle is to take advantage of the resistance of 

 the spores of the bacillus to heat. A sloped tube of in- 

 spissated serum or a deep tube of glucose agar is inoculated 

 with the pus and incubated at 37 C. for forty-eight hours, 

 at the end of which time numerous spore-bearing bacilli 

 can often be observed microscopically. The culture is then 

 kept at 80 C. for from three-quarters to one hour, with the 

 view of killing all organisms except those which have spored. 

 A loopful is then added to glucose gelatine, and roll-tube 

 cultures are made in the usual way and kept in an atmo- 

 sphere of hydrogen at 22 C. ; after five days the plates are 

 ready for examination. Kitasato compares the colonies in 

 gelatine plates to those of the B. subtilis. They consist of 

 a thick centre with shoots radiating out on all sides. They 

 liquefy the gelatine more slowly than the B. subtilis. 

 This method of isolation is not always successful, partly 

 because along with the tetanus bacilli, both in its natural 

 habitats outside the body and in the pus of wounds, other 

 spore -form ing obligatory and facultative anaerobes occur, 

 which grow faster than the tetanus bacillus, and thus over- 

 grow it. 



(2) If in any discharge the spore-bearing tetanus bacilli 

 be seen on microscopic examination, then a method of 



