the supernatant broth is syphoned off, leaving about 15 c.c. out 

 of 100 c.c., in which the deposit is re -suspended. Rabbits receive 

 5 c.c. intravenously for each injection; the injections are. given 

 in series on 2 or 3 consecutive days and at weekly intervals, and 

 after about 2-3 months a usable serum is often produced. 



For use as a culture medium the serum is distributed in small 

 tubes in quantities of 0*5 c.c, either neat or diluted as desired. 

 A few drops of a thick suspension of red cells (rabbit) are added 

 to favour growth and serve as an indicator. 



Cultivation of Pneumococci in Immune Serum. 



Several serological types of virulent pneumococci have been 

 grown in immune serum, though the majority of the observations 

 recorded have been made upon stock strains of Types I. and II., 

 which were obtained from the Rockfeller Institute. The action of 

 the homologous serum upon a pneumococcus culture has been 

 controlled by simultaneous experiments with heterologous sera. 

 After each incubation at 37° C. overnight, the serum cultures 

 were sown on plates of blood agar so as to obtain discrete colonies. 

 With very few exceptions, which will be referred to later, no 

 alteration was observed in the characters of the colonies obtained 

 after growth in a heterologous serum, and smear preparations 

 showed that no agglutination had taken place, the pneumococci 

 being distributed uniformly throughout the medium. The 

 possibility that heterologous sera produced some biological 

 modification not associated with morphological change has not 

 been investigated. In the homologous serum growth took place 

 in the form of clumps and long chains or sometimes as translucent 

 gelatinous balls which deposited at the bottom of the tube. When 

 a first culture in serum was shaken and plated, in addition to the 

 normal S type of colony, a variable number of colonies appeared 

 which differed from them in size, appearance and consistency. 

 These were the R form. A second serum culture made from the 

 first yielded, when plated, a larger number of variants from the 

 normal type. After a third passage in serum the normal S form 

 was replaced by the R. Generally the pneumococci at this stage 

 had ceased to clump, and were tending to grow uniformly through- 

 out the medium. When a loopful of the first culture in serum, 

 which, as stated above, usually consists, after a night's incubation 

 of a mixture of R and S forms, is sown into plain blood broth, 

 serial cultures up to the 13th generation have remained a mixture, 

 the normal type in this instance showing a tendency to decrease 

 in numbers relative to the R type. 



A single colony culture of either type, made at any stage of the 

 treatment with serum, as a rule, reproduces only the same type 

 when sown into plain blood broth. This rule is not without 

 exceptions, and occasionally, when the single colony culture of 

 the variant has been made after a single serum passage, a 

 reversion of some of the colonies to the normal smooth type 

 has been observed. 



* 20179 B 3 



