ISOLATION OF THE TETANUS BACILLUS 549 



The examination of the dead bodies of man or the lower animals for the 

 tetanus bacillus should be strictly limited to the site of the infection. 



It has already been pointed out that the bacillus only exceptionally becomes 

 generalized in the tissues of inoculated animals. In man, cases have been recorded 

 in which the bacillus has been found in the blood and also, but very rarely, in the 

 lymphatic glands at a distance from the infected wound. 



The investigation should take the following lines : 



(a) Microscopical examination. Examine the pus or membranous material 

 from the wound in films stained with carbol-crystal-violet or dilute carbol- 

 fuchsin and by Gram's method. 



It is often necessary to prepare several films before the bacillus can be found, as 

 it occurs in very small numbers and moreover its presence may be masked by the 

 .large number of other organisms present. Occasionally, the reverse is the case and 

 the tetanus bacillus occurs hi fairly large numbers while other organisms are scanty. 

 Fig. 259 is a reproduction of a film of pus from a case of this kind in which the 

 presence of the bacillus was easily and rapidly demonstrated. 



If microscopical examination fail to reveal the bacillus there is no justifica- 

 tion for concluding that the organism is not present ; cultures must then 

 be sown with the material. 



(6) Cultures. Isolation. Kitasato was the first to devise a means of 

 isolating the tetanus bacillus in pure culture from pus containing the organism. 

 The method is based upon the resistance of the spore to heat and upon the 

 anaerobic properties of the bacillus, and is as follows : 



1. Sow the pus or other material from the infected wound in beef broth 

 and incubate in vacuo at 38 C. 



2. After 5 days the broth now cloudy contains together with other anaerobic 

 bacteria numerous pin-shaped [drum-stick] bacilli, which are readily identified 

 as tetanus bacilli. In order to isolate the organism, pipette a little of the 

 impure culture into a fine tube, such as the narrow part of a Pasteur pipette, 

 seal the tube at both ends in the flame and heat at 100 C. for 1 or 2 minutes. 

 The spores of the tetanus bacillus survive while most of the other organisms 

 are killed. 



3. Sow the contents of the heated tube in broth and incubate in vacuo : 

 in the resulting growth the tetanus bacillus predominates and may even 

 be in pure culture. By thus alternately heating and cultivating two or three 

 times the bacillus can be isolated in pure culture. 



4. Still, it often happens that the tetanus bacillus cannot be altogether 

 freed from the bacillus of malignant oedema and another anaerobic bacillus 

 which is not pathogenic and the spore of which is not strictly terminally 

 situated. In a case such as this the experiment will have to be completed 

 by isolation in a Vignal's or Veillon's tube (p. 103). 



(c) Experimental inoculation. Inoculate a little of the pus or a small 

 fragment from the infected wound directly into guinea-pigs or mice. Symp- 

 toms of tetanus soon follow the inoculation. Cultures also should be tested 

 by inoculation into animals. 



Bacillus botulinus. 1 



The Bacillus botulinus was isolated by van Ermengem from some ham the 

 consumption of which at Ellezelles in December 1895 was followed by the 

 symptoms of botulism 2 in a number of persons who had partaken of it and 

 of whom three died. 



Homer investigated a similar epidemic at Alsfeld and isolated an organism prac- 

 tically identical with van Ermengem' s bacillus. 



1 This section has been added. 2 Botulus, a sausage. 



