CULTIVATION AND METABOLISM 931 



Cultivation and Metabolism. — None of the pathogenic species has yet been 

 cultivated apart from living cells. Of the commensal species, R. melophagi, found 

 in the sheep-ked, is said to have been grown on blood agar. Noguchi (1926) claimed 

 to have cultivated some of the commensal rickettsiae, found in ticks, on leptospiral 

 medium containing 0-2 per cent, of a carbohydrate ; when 1 per cent, of agar 

 was added, and the medium was slanted, surface colonies were obtained. Working 

 with the rickettsiae from Kocky Mountain fever and from typhus, Wolbach and 

 Schlesinger (1923-24) succeeded in obtaining growth in tissue cultures. The 

 organisms survived and multiplied only in the endothelial cells. Primary cultures 

 remained alive and virulent for 1 to 2 weeks as a rule, and later-generation cultures 

 for 2 to 4 weeks. Nigg and Landsteiner (1930) showed that cultivation of the 

 typhus virus could be accomplished in a medium, similar to that described by 

 Maitland and Maitland (1928) for vaccinia virus, which contains living but not 

 actively proliferating cells. A study by Zinsser and Schoenbach (1937) of the 

 growth of rickettsiae in such a medium showed that multiplication of these organisms 

 did not begin till after the tissue cells had ceased growing actively. This was 

 different from the behaviour of viruses, which multiply most during the stage 

 of tissue cell growth. Burnet (1938) has made similar observations, and would 

 regard this difference in metabolism between the rickettsiae and filtrable viruses 

 as of classificatory value. Another method used successfully in the cultivation 

 of the filtrable viruses, namely growth on the chorio-allantoic membrane of the 

 developing chick embryo, was found by da Cunha (1934) to be applicable to 

 rickettsiae. An even more successful method is that described by Cox (1941) of 

 growing rickettsiae in the yolk sac of the developing chick embryo. Pure, or 

 practically pure, strains of R. prowazeki may be obtained by Weigl's method of 

 intrarectal injection of body lice with infective material. The intestine of the 

 louse is practically free from ordinary bacteria, so that it serves as an almost sterile 

 medium for the cultivation of rickettsiae. In practice, in vivo cultivation in the 

 tissues of a susceptible animal, such as the testicle of the guinea-pig or rabbit, 

 is frequently employed for preserving strains of typhus virus ; according to 

 Kodama and Takahashi (1931), viruses kept in this way undergo no change in 

 antigenic structure or pathogenicity. Certain rickettsial strains, such as R. nip- 

 ponica, can be cultivated in the anterior chamber of the rabbit's eye (Nagayo 

 et al. 1930). After an incubation period of 4 to 8 days iritis develops, similar 

 to that occurring naturally in tsutsugamushi fever. Histological examination 

 reveals the presence of peculiar corpuscles in the endothelial cells of Descemet's 

 membrane, consisting apparently of colonies of rickettsiae. The animal method 

 of cultivation has been applied particularly to R. prowazeki and R. mooseri in an 

 effort to provide heavy suspensions of rickettsiae for serological and vaccination 

 purposes. The mouse's lung has proved particularly useful for this purpose, 

 the animals being inoculated intranasally (Okamoto 1937, Castaneda 1939, Durand 

 and Sparrow 1940, Giroud and Panthier 1942). 



Little is yet known about the growth requirements of Rickettsia, but interesting 

 observations have been made. Pinkerton and Hass (1932) found that in plasma 

 tissue cultures R. prowazeki grew best at 32° C. This behaviour may be related 

 to the preference that some species of rickettsiee show, after inoculation into the 

 peritoneal cavity of the guinea-pig, for growth in the scrotal sac, where the tem- 

 perature is lower, rather than for the general peritoneal cavity. In the Maitland 

 medium growth occurs equally well at 32° C. and at 37° C. This apparent dis- 



