Serological Differentiation q/ Bacillus tetani. 147 



the addition of fresh rabbit tissue. Prepared in this way, the medium inhibits 

 the growth of B. sporogenes, and allows of the growth of B. tetani, atoxic 

 tetanoid bacilli, and oval-endsporing organisms. The value of this medium, 

 in its application to the study of B. tetani, will be considered later. 



III. Application of Serological Methods to the Sticchj of Toxic Cultures. 



Using this putrid medium and Barber's micro-inoculation method, I 

 succeeded, with the assistance of Miss Eobertson, of the Lister Institute of 

 Preventive Medicine, in isolating from wounds a small number of strains of 

 B. tetani in a fair state of purity. 



Particular attention is called to the question of purity of cultures of the 

 anaerobes. I advisedly describe the growths which are under consideration 

 as being only relatively pure, since extreme difficulty is experienced in 

 completely purifying these cultures. This difficulty is due probably to the 

 symbiotic relationship which the anaerobes bear to one another. 



I would here record my deep debt of gratitude to Miss Eobertson for her 

 kindness in purifying these growths for me, as they formed the basis for 

 serological methods being applied to the examination of the question under 

 consideration. 



Having obtained from wounds a number of fairly pure growths known to 

 be toxic, I immunised a rabbit against one of the laboratory strains of 

 B. tetani, with a view to the production of an agglutinating serum. 



The strain of B. tetani chosen was an isolation made by Miss Eobertson 

 from the U.S.A. standard culture. This culture will hereafter be designated 

 " A." The process of immunising the animals was as follows : — 



A culture of the organism to be inoculated was grown in peptone broth 

 neutral to a-naphtholphthalein under anaerobic conditions at 37° C. for a 

 period of four days. ' The cultures were then heated to 60° C. for 30 minutes, 

 and thereafter centrifugalised at high speed to deposit the organisms. The 

 supernatant fluid was pipetted off, saline added, and the suspension so 

 obtained was again centrifugalised. 



The deposit resulting from this second centrifugalisation was shaken in a 

 small quantity of saline, and the resulting suspension was standardised. 

 Sufficient saline, containing 0-5 per cent, plienol, was then added to reduce 

 the bacillary content of the suspension to 2000 million per cubic centimetre. 



In immunising the animals, 0'7o c.c. of this suspension is inoculated 

 intravenously, and, after an interval of five days, I'o c.c. is injected by the 

 same route ; after a further lapse of two days, the agglutinating titre of the 

 serum is tested, and, if suitable, the animal is killed. No difficulty was 

 experienced in obtaining a serum of moderate titre — 1 /800 in two hours at 



