36 



though it cannot be reproduced in vitro. It occurred to me that 

 some information might be obtained on this point by the 

 examination of strains grown from sputum during the acute 

 stage of pneumonia and later during convalescence. If mutation 

 occurred, one might expect some regularity in the serological 

 characters of the strain which replaced the Types I and II. 

 A number of experiments have been made on these lines in 

 infections with Types I and II and I have made it the routine 

 to test several colonies from each case, obtained either through 

 the mouse or direct from the sputum. Although many of the 

 infections have been apparently pure, it is very striking how 

 often atypical bile-soluble diplococci can be found in sputum, 

 even during the acute stage, together with the Types I and II 

 to which must be attributed the production of the pneumonia. 

 Before proceeding to describe some of these results, it may be 

 useful to indicate the method adopted for investigating single 

 colony cultures. 



Method of obtaining Single Colony Cultures. 



At first some difficulty was experienced in obtaining uniformly 

 successful growth of colonies of pneumococci in broth. The 

 following procedure was ultimately adopted. The material, 

 sputum or mouse blood, was plated on nutrient agar, to which 

 was added 5 per cent, of chloroformed blood cells from horse 

 or ox with 5 per cent, filtered horse or bovine serum. This 

 makes an opaque medium on which pneumococci grow well and 

 form characteristic colonies, so that it was rare to select a colony 

 which failed to pass the bile solubility test. The selected colony 

 was touched with the point of a spatula and rubbed up in a small 

 tube containing about Oo c.c. of 50 per cent, whipped rabbit 

 blood in broth. After incubation overnight, a loopful of the 

 deposited cells, dark in colour where growth of pneumococci had 

 occurred, was transferred to a tube containing about 5 c.c. of 

 trypsinised broth. After a night's incubation a peptone broth 

 culture was inoculated from the tryp. broth (the latter being 

 unsuitable for the bile solubility test), the remainder of which 

 was used for test with agglutinating type sera. 



Details of some Cases from which Mixed Cultures were obtained. 



The following are a few examples of cases of pneumonia in 

 which the examination of the sputum revealed the presence of 

 more than one type of pneumococcus. It has been observed 

 that a second type has more frequentlv been obtained in the 

 B where the primary infection was due to T. I than T. II. 

 In no instance have both Types I and II been obtained from the 

 same sputum. The second type has always belonged to Group 

 IV, and it is of some interest to note that, where it has been 

 possible to identify a homologue of the 12 types of Group IV, 



