224 BACTERIA PATHOGENIC TO MAN 



Isolation of Pure Cultures. The growth of the tetanus bacillus in the 

 animal body is comparatively scanty, and is usually associated with 

 that of other bacteria; hence, the organism is difficult to obtain in pure 

 culture. The method of procedure proposed by Kitasato, which, how- 

 ever, is not always successful, consists in inoculating slightly alkaline 

 nutrient agar or glucose bouillon with the tetanus-bearing material 

 (pus or tissue from the inoculation wound), keeping the culture under 

 anaerobic conditions for twenty-four to forty-eight hours at a tempera- 

 ture of 37 C., and, after the tetanus spores have formed, heating it 

 for one-half an hour at 80 C., to destroy the associated bacteria. 

 The spores of the tetanus bacillus are able to survive this exposure, 

 so that when anaerobic cultures are then made in the usual way the 

 tetanus colonies develop. When the tetanus bacilli are the only spore- 

 bearing bacteria present, pure cultures are readily obtained ; when other 

 spore-bearing anaerobic bacteria are present, the isolation of a pure 

 culture may be a matter of difficulty. 



Pathogenesis. In mice, guinea-pigs, rabbits, horses, goats, and a 

 number of other animals inoculations of pure cultures of the tetanus 

 bacillus cause typical tetanus after an incubation of from one to three 

 days. A mere trace of an old culture only as much as remains cling- 

 ing to a platinum needle is often sufficient to kill very susceptible 

 animals like mice and guinea-pigs. Other animals require a larger 

 amount. Rats and birds are but little susceptible, and fowls scarcely 

 at all. Man is more susceptible than any of the animals so far tested. 

 A horse is about six times as sensitive as a guinea-pig and three 

 hundred thousand times as sensitive as a hen. It is a remarkable fact 

 that an amount of toxin sufficient to kill a hen would suffice to kill 500 

 horses. It is estimated that if 1 gram of horse requires 1 part of toxin 

 to kill, then 1 gram of guiriea-pig requires 6 parts, 1 of mouse 12, of 

 goat 24, of dog 500, of rabbit 1500, of cat 6000, of hen 360,000. 

 Cultures from different cases vary greatly in their toxicity. On the 

 inoculation of less than a fatal dose in test animals a local tetanus 

 may be produced, which lasts for days and wrecks and then ends in 

 recovery. On killing the animal there is found at autopsy, just at the 

 point of inoculation, a hemorrhagic spot, and no changes other than 

 these here or in the interior organs. A few tetanus bacilli may be detected 

 locally with great difficulty, often none at all; possibly a few may be 

 found in the region of the lymphatic glands. From this scanty occur- 

 rence of bacilli the conclusion has been reached that the bacilli of 

 tetanus, when inoculated in pure culture, do not multiply to any great 

 extent in the living body, but only produce lesions through the absorp- 

 tion of the poison which they develop at the point of infection. It has 

 been found that pure cultures of tetanus, after the germs have sporulated 

 and the toxins been destroyed by heat, can be injected into animals 

 without producing tetanus. But if a culture of non-pathogenic organ- 

 isms is injected simultaneously with the spores, or if there is an effusion 

 of blood at the point of injection, or if there was a previous bruising 

 of the tissues, the animals surely die of tetanus. Even irritating foreign 



