586 THE PNEUMOCOCCUS 



Another cause of the attenuation and death of the pneumococcus in culture is 

 the rapidity and extent to which it forms acid (for the most part formic acid). The 

 addition of calcium carbonate to the medium neutralizes the acid as it is formed, 

 and in this way the vitality of the organism can be preserved for more than a month 

 (Wurtz and Mosny). According to Bolduan, the calcium salt maintains the life 

 of the culture not because it neutralizes the acid but on account of a special action 

 of the calcium on the pneumococcus. 



Restitution and exaltation of virulence. (a) The virulence of an attenuated 

 pneumococcus can be restored by inoculating into a rabbit fairly large doses 

 (1 c.c.) of a broth culture of the organism and at the same time an equal 

 quantity of a filtered culture of Proteus vulgaris. The animal will die from a 

 pneumococcal septicaemia and the organism in the blood will be found to be 

 virulent. 



(6) The virulence of the pneumococcus can be increased by passages through 

 rabbits. Intra-venous inoculation is better for this purpose than sub- 

 cutaneous, but intra -peritoneal inoculation is a more certain method than 

 either (Issaeff). 



Inoculate 1 or 1*5 c.c. of the blood of a rabbit dead of a pneumococcal septicaemia 

 into the peritoneum of a rabbit (A). Inoculate a second rabbit with the blood of 

 A and so on in series until eight or nine rabbits have been inoculated. The amount 

 of blood inoculated is at this stage diminished ; for the eleventh rabbit, for 

 instance, 6 or 8 drops of the virus are sufficient. 



From about the twelfth passage onward the blood loses its power of coagulation, 

 becomes extremely toxic and virulent and contains very numerous pneumococci. 

 One drop of this blood inoculated into the peritoneum of a rabbit will prove fatal 

 in 10 or 12 hours : if too large a dose (for example 12 c.c.) be given the rabbit will 

 die very quickly (5 or 6 hours) of toxaemia and not of septicaemia. 



Sub-cutaneous inoculation of 4 or 6 drops of blood containing a pneumo- 

 coccus the virulence of which has been increased in this way will kill rabbits 

 in 12 or 15 hours. It is to be noted however that after a long series of passages 

 through rabbits the virulence of the organism becomes attenuated but can 

 be restored by two or three passages through another species such as the 

 guinea-pig or dog. 



2. Bio-chemical reactions, 



The bio-chemical reactions of the pneumococcus and the characteristics 

 which distinguish this organism from the streptococci are described on 

 p. 602. 



3. Toxins. 



(i) Filtered cultures of the pneumococcus are only slightly toxic. If 

 inoculated in large quantities into the veins of rabbits they give rise to a 

 transitory rise of temperature and loss of weight : death does not generally 

 result. 



A somewhat more toxic product is obtained by sterilizing cultures of the 

 organism by chloroform or heat (a temperature of 58 C. for 2 hours will 

 sterilize the culture without altering the toxin). Cultures on young rabbit- 

 serum yield the most powerful toxin. 



Anaerobic cultures in broth or serum which are kept alkaline offer no 

 advantage from the point of view of toxin production. 



The brothers Klemperer isolated a toxin by precipitating filtered broth cultures 

 with alcohol or sulphate of ammonia : Foa and Carbone obtained similar results. 

 Andreini attributed the toxicity of cultures to an alkaloidal base. 



(ii) Emmerich obtained a more toxic product by crushing and expressing 

 the organs of rabbits which had died of a pneumococcal septicaemia and 

 filtering the juice through a bougie. Mosny modified this method as follows : 



Immediately after death the organs of the rabbit are minced and macerated in 



