BACILLUS TETANI 511 
(1 to lOOO) kills them in about three hours. The addition of O.o per 
cent hydrochloric acid increases the germicidal action of both carbohc 
acid and mercuric chloride. A 1 per cent solution of silver nitrate 
kills tetanus spores in one minute, and a 0.1 per cent solution in ten 
minutes. Iodoform is said to be very effective in preventing the ger- 
mination of these spores. 
Tetanus spores are very resistant to drying. Kept in a cool, dark 
place they may remain viable for years. Henrijean^ has found that 
they may remain alive and produce virulent cultures after a period of 
nearly eleven years. 
Conditions of Growth.— B. tetani is an obligate anaerobe, but its 
tolerance to oxygen is somewhat greater than that of many other 
members of the anaerobic group. The development of the organism, 
however, is best in the oxygen-free environment, and it is only under 
such conditions that the full toxicogenic powers of the bacillus are 
realized.^ An atmosphere of hydrogen, of nitrogen, or a vacuum, 
creates a condition favorable to the production of toxin. An atmos- 
phere of carbon dioxide, however, is said to be unfavorable to growth. 
Growth ceases below 14° C. and above 44° C, the optimum being 37° C. 
Growth at 20° C. is sluggish, and sporulation proceeds slowly. Sporu- 
lation is maximal at 37° C, and is seriously interfered with if the tem- 
perature exceeds 42° C. 
Isolation and Culture.— Pure cultures of tetanus bacilli are difficult to 
obtain, either from the soil, or from other sources where they exist in 
association with other bacteria. Kitasato^ succeeded in isolating the 
organism by incubating slightly alkaline broth, containing tetanus 
and other bacilli, for forty-eight hours at 37° C, then heating the 
medium to 80° C. for thirty minutes to destroy vegetative forms. 
Successive repetitions finally resulted in the growth of a pure strain. 
This procedure is not ordinarily feasible, however. Theobald Smith^ 
has devised a method of obtaining pure cultures of spore-forming 
anaerobes, including tetanus bacilli, which is far more successful in 
practice than the Kitasato^ method. I^nrichment and acclimatization 
of the organisms is obtained in fermentation tubes containing bits of 
sterile tissue. After an incubation at 37° C. for forty-eight hours the 
culture is heated to 80° C\ for thirty minutes to kill vegetative forms. 
Material from the sediment around the piece of tissue is reinoculated 
into another fermentation tube, heated, and plates are made from the 
residue, which contains only viable spores. Blood-agar plates, incu- 
bated anaerobically, are well adapted for plating. Tulloch has 
made the interesting observation that tetanus bacilli grow upward 
rapidly upon agar slant cultures in long filaments. If a mixed culture, 
1 Ann. de la Soc. med.-chir. de Li^ge, 1891, No. 10, p. 367. 
2 Ferran: Centralbl. f. Bakteriol., 1898, 24, 28. 
* Loc. cit. 
i Jour. Boston Soc. Med. Sci., June 20, 1S99; Jour. Med. Res., 1905, 14, 193. 
= Loc. cit. 
