•till viable; on plating, some yielded apparently all smooth 

 colonies, and, where rough colonies were detected, they were few 

 in number. A rough colony culture, which has been produced 

 from a virulent strain by a single passage in immune serum, may 

 retain its rough characters for many generations in plain blood 

 broth and, as already mentioned, may subsequently revert to the 

 smooth type. This has occurred particularly with Type II., and 

 the reversion in cultural characters has been associated with the 

 recovery of virulence. Certain of the R colony strains, which 

 have been subcultivated for many generations in plain blood broth 

 and have been plated at intervals, have become in appearance 

 almost indistinguishable from the S strain, but they have 

 remained attenuated. 



The change in the biological characters is of greater importance 

 than the morphological differences between the colonies. On the 

 other hand the characteristics of the R type obtained by three 

 passages through immune serum have been maintained for more 

 than 13 subsequent generations in plain blood broth. The activity 

 of the immune serum, i.e., the height of its titre, appears to have 

 an influence upon the permanence of the change. 



Vibulence. 



So far as my observations go at present the typical S colony 

 derived from a virulent culture is always pathogenic for mice 

 though one cannot affirm that all S colonies are equally virulent. 

 There is some evidence that a partial degree of attenuation is 

 associated with a corresponding tendency to the rough state, and 

 such partially rough colonies yield typically smooth colonies after 

 causing septicaemia in a mouse. The typical R colony shows, on 

 the other hand, a high degree of attenuation. When a virulent 

 pneumococcus is grown overnight in immune serum, a plate made 

 from the serum culture yields a mixture of R and S colonies. If 

 a colony of each type is selected and subcultivated for several 

 generations in plain blood broth, the S colony culture is found to 

 be as virulent as the original culture, e.g., killing mice in doses of 

 0-000,000,1 c.c. and 0*000,000,01 c.c. of broth culture, while the 

 R culture has no effect in doses of 0* 1 c.c. and 0* 2 c.c. (the latter 

 being the greatest amount inoculated). 



Retested on mice after 18 generations in blood broth, the above 

 cultures gave similar results : the R culture failed to kill mice in 

 doses ranging up to 0*25 c.c. of blood broth culture, while the 

 S culture had remained apparently unaltered in virulence. If a 

 mouse dies of typical septicaemia after an inoculation of an appar- 

 ently typical R culture, the colonies grown from the blood are 

 invariably of the S type, that is, the culture has reverted. Occa- 

 sionally, however, a mouse may die after a large dose of R culture, 

 and the only organisms recoverable from the blood are a few 

 colonies of the R type. In such a case the peritoneal washings 

 do not give the characteristic agglutino-precipitin reaction with 

 the type serum prepared from the normal strain, and the blood of 



