22 



of the mouse, preserved by Heim's method, provided a reserve 

 in the case of each strain. 



Preparation of Pneumococcal Agglutinating Sera in Babbits. 



Adult rabbits were immunised with nscending doses of pneumococci 

 which at first wore killed by heat and finally were used living. The 

 following are the details of n successful immunisation experiment with 

 the American Type III strain on a rabbit, the serum of which developed 

 an unusually high agglutination tit re and protective value. The prelim- 

 inary treatment with killed culture, i.e., the centrifuged deposit of 

 trypeinised broth culture heated to 60° C for i hour, lasted for three 

 weeks, during which the animal received three series of intravenous 

 inoculation-. After an interval of six days immunisation was continued 

 with living culture. This was first administered subcutaneously in a 

 series of inoculations on four successive days followed, after a rest of 

 i days, by a second series of three inoculations. The third series was 

 given intravenously, beginning with a dose of 0-5 c.c. of broth culture. 

 The dose was gradually increased, the rabbit finally receiving the deposit 

 of 20 c.c. of virulent broth culture. After 10 days from the last injection 

 the rabbit was bled, the total period of treatment being 9 weeks. The 

 fellow rabbit to the above received identical treatment, but it never 

 developed a usable serum though immunisation was prolonged for more 

 than twelve months. Probably peculiarities in individual rabbits are 

 more important than the method of inoculation; some rabbits appear to 

 be incapable of producing a good antiserum. As regards the condition 

 of the antigen the use of living cultures guards against unfavourable 

 changes. There is, however, the disadvantage that in spite of a high 

 content of protective substances in the serum, induced by the preliminary 

 inoculal ions of killed culture, the animals may die suddenly from endocard- 

 itis. large vegetations being found post-mortem on the mitral valve cusps. 

 The risk may be avoided, since, as I have found more recently, active 

 agglutinating and protective sera can be obtained from rabbits inoculated 

 solely with culture killed by heat. Probably the maintenance of the 

 strain in a condition of maximum virulence is an important factor in the 

 production of a good immune serum, the fact of its being used living or 

 dead being subsidiary. On this hypothesis every effort has been made 

 to maintain the original- virulence of those strains which have been used 

 for the preparation of sera. The stock cultures have been passed through 

 mice every two or three weeks and the culture in blood broth made direct 

 from the mouse's blood has been kept on ice after a single night's incubation 

 at 37° C. 



Agglutinating sera of a sufficiently high titre for diagnostic purposes 

 have been produced after two to six months treatment ; the titre of these 

 sera has ranged from 1 in 40 to 1 in 640 and occasionally has been as high 

 a- 1 in 1,280. In the case of certain strains it was very difficult to 

 obtain any but relatively low titred sera. 



Method of Performing Agglutination Test. 

 At the beginning of this investigation I used for agglutination 

 tests pneumococci obtained by centrifuging glucose broth cul- 

 tures and suspending the deposit in salt solution. Such sus- 

 pensions, in my experience, are often unstable and may show 

 some clumping with sera prepared with heterologous types. The 

 tendency towards auto-agglutination is increased if the glucose 

 broth cultures are incubated long enough for autolysis of the 

 cocci to begin. Growth in the above medium should not be 

 allowed to proceed beyond the stage where the broth culture is 



