588 THE PNEUMOCOCCUS 



(ii) With attenuated cultures. Animals can be immunized by inoculating 

 them with living cultures the virulence of which is attenuated by age. Thus, 

 for example, broth cultures 5 or 6 days old are used for the first inoculation, 

 and the immunization is continued by using younger and younger cultures, 

 until finally a 24-hour growth, or a dose of virulent blood, is inoculated (Foa 

 and Scabia, Netter, Washbourn). 



(iii) With stained cultures. Sergent sows agar cultures with the blood of 

 a rabbit which has died of a highly virulent pneumococcal infection. After 

 incubation the growth is scraped off and made into an emulsion with sterile 

 normal saline solution containing a few drops of a sterile aqueous solution 

 of crystal-violet. After about an hour all the organisms are stained but are 

 still living and on sub-culture give rise to a new culture. A volume of this 

 stained emulsion equivalent to one-tenth of an agar culture will kill rabbits 

 in 12-48 hours when inoculated sub-cutaneously, but it produces no effect if 

 inoculated intra-venously or intra-peritoneally. Rabbits which have received 

 several of these harmless intra-venous or intra-peritoneal inoculations at 

 intervals of &-8 days (the dose of emulsion inoculated may be progressively 

 increased) are able to resist the inoculation of a quantity of a virulent culture 

 which is sufficient to kill a normal animal in less than 24 hours. These rabbits 

 are more highly immunized than animals vaccinated with sterilized cultures. 



(iv) With bacteriolyzed cultures. Neufeld found that if 0- 1-0-2 c.c. of 

 rabbit bile was added to 2 c.c. of a 24-hour-old broth culture of the 

 pneumococcus the culture became clear in 15-20 minutes : the cocci were 

 killed and bacteriolyzed and disappeared entirely. The same result is obtained 

 by using a 5 per cent, solution of cholate or taurocholate of sodium instead 

 of bile. Other micro-organisms, and especially the pyogenic streptococci, 

 are not destroyed under these conditions. 



Neufeld, Nicolle and Adil Bey, have shown that rabbits can be highly 

 immunized by a single inoculation of 2 c.c. of a culture bacteriolyzed as 

 above. 



5. Serum therapy. 



(i) The blood of animals naturally immune to the pneumococcus has 

 neither therapeutic nor immunizing properties. 



(ii) The blood of vaccinated animals is not antitoxic and is incapable of 

 neutralizing the toxins of the pneumococcus either in vitro or in vivo (Issaeff). 



(iii) The serum of vaccinated animals has no bactericidal action on the 

 pneumococcus in vitro. The pneumococcus will grow in the serum but 

 somewhat poorly and will retain its virulence though it undergoes some 

 morphological modifications the rounded forms predominate and the 

 micro-organisms are agglutinated in clumps (Behring and Nissen, Issaeff). 



In verifying the virulence of a pneumococcus grown in the serum of a vaccinated 

 animal it is necessary to exclude a source of error which may arise from inoculation 

 of the entire culture. The culture is composed of two parts : (1) the micro-organisms 

 and (2) the serum which serves as the culture medium and which, as will be seen 

 immediately, has the power of conferring immunity. If the whole culture be 

 inoculated the animal being immunized by the serum inoculated with the organisms 

 will survive the inoculation, and the erroneous conclusion will be that the serum 

 is bactericidal. One or other of the following devices must therefore be adopted. 



(a) Sow a trace of the serum culture into a tube of broth and inoculate the broth 

 culture. This method is open to criticism. 



(b) Pour the serum culture on a sterilized filter paper. Wash the organisms two 

 or three times with sterile normal saline solution. Collect the residue with a brush, 

 dilute it in 2 c.c. of a normal saline solution and inoculate the emulsion sub- 

 cutaneously. 



(iv) The serum of vaccinated animals has both prophylactic and thera- 



