indistinguishable in appearance, there seems to me little doubt 

 that both he and Arkwright are dealing with essentially the 

 same phenomenon and that the differences in some of the results 

 of the two workers merely represent different stages in the 

 tendency of a culture to separate into individuals with distinc- 

 tive characters. 



The varieties of pneumococcus colonies which I am about 

 to describe show differences in morphology and in some of their 

 immunological reactions, which are similar to those found by 

 Arkwright in other bacterial species. They were discovered 

 in the course of my investigation of the mode of action of anti- 

 pneumococcus serum. On plating cultures of pneumococci 

 grown on homologous antisera, it was found that a number of 

 colonies which differed from the normal in appearance were 

 attenuated in virulence for mice.* 



Attenuation was definitely associated with the production of 

 rough colonies, and these could be clearly distinguished from those 

 of smooth appearance which retained their virulence. 



Culture Media. 



Plate cultures. — Nutrient agar prepared with trypsinised meat 

 was used, with the addition of 5 per cent, chloroformed whipped 

 horse blood and 5 per cent, filtered horse serum. On this medium 

 isolated colonies of pneumococci grow up to 2 mm. in diameter, 

 and autolysis is often very slight. 



The horse blood should be collected from a vessel directly into 

 a sterile bottle, since chloroform fails to get rid of gross contami- 

 nation. The blood cells, which rapidly deposit, are syphoned off 

 into medicine bottles, and about 2 per cent, chloroform is added : 

 it is useful to have some glass beads in the bottle to break up the 

 cells, which form a thick mass under the action of the chloroform. 

 The horse serum is filtered through a Berkefeld candle. 



Blood broth. — Colonies from plates are grown in small tubes, 

 3 inches by \ inch, containing about 1 c.c. of equal parts of 

 whipped rabbit blood and trypsinised meat broth. Growth occurs 

 rapidly in the small bulk of medium, and can be recognised by 

 the medium becoming dark purplish in colour. The blood cells 

 keep almost intact in the ice chest for several weeks, and are 

 useful in showing the presence or absence of haemolysis due to 

 bacterial growth. After inoculation and incubation the cells 

 fall to the bottom of the tube, and a loopful of the deposit always 

 produces a good subculture of pneumococci in a tube containing 

 about 6 c.c. of ordinary broth. 



Immune Serum. — The sera are prepared in rabbits from 

 virulent cultures grown for 4-6 hours in glucose broth. The 

 broth cultures are heated to 60° C. for an hour and centrifuged ; 



* Laura Stryker (Joum. Exp. Med., xxiv, p. 49, 1916) had already 

 observed that repeated subculture of Types I. and II. pneumococci in 

 homologous serum caused marked reduction in virulence. 



