BACILLUS TETANI 473 



Isolation and Culture. Pure cultures of tetanus bacilli are difficult 

 to obtain from the soil or from other sources, where it exists in asso- 

 ciation with other bacteria. Kitasato 1 succeeded in isolating pure 

 cultures by alternately incubating alkaline broth containing tetanus 

 and other bacteria anaerobically at 37 C. for forty-eight hours, then 

 heating the culture to 80 C. for thirty minutes to destroy non -spore- 

 forming organisms. Theobald Smith 2 has devised a method for obtain- 

 ing pure cultures of spore-forming anaerobes, including tetanus bacilli, 

 which is far more successful in practice than the Kitasato method. 

 Fermentation tubes containing sugar-free broth slightly alkaline in 

 reaction and bits of sterile tissue (kidney or liver from rabbits or 



FTG. 65. Bacillus tetani, spore formation. X 1000. (Giinther.) 



guinea-pigs) are inoculated with the suspected material and incubated 

 at 37 C. for forty-eight hours. The growth of anaerobic organisms 

 is much more luxuriant in this tissue medium than in similar media 

 without the tissue. 3 Tetanus spores are formed abundantly around 

 the bit of tissue, and after forty-eight hours of incubation the culture 

 is again heated to 80 C. for thirty minutes to kill non-spore-forming 

 organisms. The spore-containing medium which collects at the lowest 

 part of the fermentation tube around the bit of tissue is reinoculated 

 into a fresh fermentation tube of the same medium, and the process 

 is repeated in detail. It is advisable to use a pipette to remove the 

 material for inoculation in order to insure an abundance of spores. 

 The success of the procedure is readily controlled by stained prepara- 



1 Loc. cit. 



2 Jour. Boston Soc. Med. Sci., 1899, 340; Jour. Med. Research, 1905, xiv, 193. 



3 This method has been rediscovered by Tizzoni (Centralbl. f. Bakt., Orig., 1905, 

 xxxiv, 619), and others. 



