the mouse produces no effect when inoculated into a second mouse. 

 The following example shows how repeated passage through im- 

 mune serum fixes the attenuated character. A colony of a Type 

 III. strain, virulent for mice in a dose of 0*000,000,1 c.c. of broth 

 culture, was cultivated for one night in Type III. serum ; a second 

 serum oulture was sown from the first. Both serum cultures were 

 plated when 24 hours old, and an R colony was subcultivated from 

 each in plain blood broth. (The R colonies of a Type III. strain 

 are readily distinguished from the large watery S colonies.) Each 

 culture was tested on mice. The R culture from the first serum 

 killed mice, and was found on being recovered from the blood to 

 have reverted to the S type. The colony culture from the second 

 serum was completely attenuated, and failed to kill mice in a dose 

 of 0-2 c.c. 



Antigenic Characters of R and S Cultures. 



My observations have not been numerous but they show that 

 the two types of colonies have different antigenic qualities. Sera 

 have been prepared in rabbits with the R and S cultures of both 

 Type I. and Type II. The results with Type I. sera, which I am 

 about to describe, were also obtained with the first samples of 

 Type II. sera. Later samples of the Type II. sera showed less 

 difference between the R and S sera, and on investigation it was 

 found that the R culture used for immunisation had partially 

 reverted to the S type. The Type I. sera were prepared with 

 strains which retained their special characters unaltered during 

 the period of immunisation. Three rabbits were immunised with 

 R culture, and one with S culture : the cultures were heated to 

 60° C, and were prepared freshly before each injection. All the 

 rabbits received identical treatment, 12 injections being given 

 intravenously during a period of five weeks. Samples of blood 

 were withdrawn eight days after the last inoculation. 



Agglutination. — The serum from the rabbit injected with the 

 S culture agglutinated both S and R cultures up to a dilution of 

 1 in 160. The product of the reaction between the serum and the 

 S culture consisted of firm masses, which could not be broken 

 up by shaking. The R culture agglutinated with the formation 

 of loose clumps which were readily broken up on shaking to form 

 a finely granular suspension. 



Of the three rabbits injected with R culture one only yielded 

 a good agglutinating serum. This agglutinated the homologous 

 R strain up to 1 in 640, and the clumps formed were of the same 

 loose character as those described above in the case of the reaction 

 between the R culture and the S serum. The S culture was 

 barely agglutinated in 1 in 10 with the R serum. 



Precipitin. — The sera were tested upon broth culture which 

 had been freed from cocci by centrifuging and filtration through 

 a Berkefeld candle. When the S serum was mixed with the 

 filtrate of the S oulture, a precipitate was formed which aggre- 



