146 



Dr. W. J. Tulloch. The Isolation and 



infection with a number of diflferent organisms, admittedly often closely 

 allied to one another, but susceptible of classification by serological methods, 

 it seemed of importance to determine whether, in the case of tetanus, we 

 are dealing with an infection due to one and the same organism in all cases 

 of the disease, or to a number of different though closely related bacteria. 



The differentiation of the dysentery bacilli, of the enteric group of 

 organisms, of the pneumococci, and of the meningococci, indicated that 

 examination should be made of the causal organism of tetanus by all bacterio- 

 logical, and especially by serological methods. 



II. Cultivation of B. tetani. 



The main obstacle to be overcome in studying B. tetani is the difficulty 

 experienced in isolating the organism. Owing to its living in symbiosis 

 with other bacteria that grow more vigorously in artificial media than does 

 B. tetani, special methods have had to be elaborated for the (partial) isolation 

 of that organism. 



The rationale of the technique which I use depends upon the fact that if 

 material from wounds infected with the anaerobes be inoculated into a medium 

 consisting of chopped meat and water, sterilised in the autoclave, the 

 proteolytic organisms — notably B. sporof/cnes — first appear in the culture 

 and are replaced later by organisms whicla resemble the tetanus bacillus, both 

 morphologically and, to some extent, culturally. Many of these organisms 

 are non-pathogenic, and from:the standpoint of cultural reqiiirements tetani 

 is included with them. 



After several attempts with a variety of procedures, I have so far found 

 the following method most satisfactory for my purpose : — 



Take 1 lb. of chopped meat and add 1 litre of tap-water, then boil for 

 30 minutes. Cool to 45° C, make slightly alkaline, and add trypsin as for 

 the preparation of Douglas' broth, then incubate in an open vessel at 37° C. 

 for five days, and allow to undergo natural putrefaction. 



The putrescent material so obtained is filtered through paper, made 

 neutral to phenolphthalein, and sodium formate is added to the extent of 

 1 per cent, of the total. The material is then sterilised and cleared by being 

 passed through a Berkfeld and a Doulton filter in series. The medium is 

 stored in sterile flasks under paraffin, and is syphoned off as required. It 

 keeps well, but should not be sterilised by heat. 



Before use the sterility of the medium is tested by inoculating quantities 

 of from 5 to 0"1 c.c. into tubes of meat-water medium, which are incubated 

 anaerobically for seven days. This medium, while it has desirable selective 

 properties, is not sufficiently nutrient, and must be enriched before use by 



