39 



Type I serum was treated three times with washed heated suspensions 

 of Type I culture. The resulting deposit was removed by centrifuging 

 and was washed with broth. The supernatant fluid and the washed 

 deposit were tested separately in mice : the latter still protected mice 

 against 0*01 c.c. of culture, the former failed to protect. 



A similar experiment was made with the serum of Pn. 10, a 

 Group IV strain. 



The heated deposit of broth culture was added to the serum on three 

 successive occasions, and the mixture was then centrifuged. The fluid 

 remained a little opalescent in spite of the prolonged centrifuging, but 

 after standing overnight small clumps formed which settled, leaving the 

 fluid quite clear. The deposit was washed twice and was suspended in a 

 quantity of salt solution equal to the original serum. 



The deposit and the original supernatant fluid were each tested upon 

 mice for protective properties. The deposit protected mice against 

 0*01 c.c. of culture but failed to protect against 0*1 c.c. The supernatant 

 fluid protected against 0*000 01 c.c. but not against 0*000 1 c.c. 



The experiment was repeated with the same serum and a smaller 

 amount of culture added on one occasion only. The supernatant fluid 

 protected against 0*01 c.c. of culture but not against 0*1 c.c. The deposit 

 failed to protect against - 01 c.c. but protected against 0*000 1 c.c. 



It will be seen that in the first experiment, where a large 

 amount of absorbing culture was used, more of the protective 

 substances were demonstrated in the deposit than in the super- 

 natant fluid. The converse was the case in the second experi- 

 ment, where the exhaustion of the serum was less severe, i.e., 

 more of the protective bodies remained in the supernatant fluid 

 than were taken down with the centrifuged deposit. The effect 

 of heating the deposit to 69° C. for 1 hour was tried, and it was 

 found that the protective properties were not impaired. 



Instead of culture the peritoneal washings of a guinea-pig 

 which had been inoculated intraperitoneally with living culture 

 of Type II were used to treat a Type II serum. It was thought 

 that perhaps pneumococcal aggressins might be produced, and 

 that these might neutralise the protective power of the serum. 

 In effect the results were the same as when the serum was treated 

 with culture cocci ; the protective substances went down with the 

 precipitate and still retained their active properties. 



The details of the experiments include a few points of interest. Before 

 addition to the serum the peritoneal washings were centrifuged clear, and 

 were sterilised by heating to 60° C. followed by chloroform. After the 

 mixture had stood over night, a firm gelatinous precipitate was formed. 

 This was removed by centrifuging and the supernatant fluid was treated 

 for the second time with the peritoneal washings : there was no further 

 visible reaction. 



The deposit was ground up in a mortar but it was impossible to make 

 a uniform suspension in broth, as the particles almost immediately became 

 agglutinated. An effort was made to inoculate mice with equal parts of 

 the deposit by dividing it up into small pieces about the same size. Five 

 mice were thus inoculated intraperitoneally, and test doses of a Type II 

 culture, virulent for mice in doses of 0*000 000 01 c.c, were given. All five 

 mice remained alive after doses ranging to 0*000 01 c.c. The supernatant 

 fluid was still protective against 0*000 01 c.c. (1 out of 2 mice). 



This experiment was repeated with Type II serum and peritoneal 

 washings with similar results. Before treatment, the Type II serum in 

 doses of 0*2 c.c. protected against * 1 c.c. of Type II culture. The 

 gelatinous deposit, representing approximately * 2 c.c. of serum, protected 

 against 0*01 c.c. of culture. 



B 4 



