BINUCLEATA 47 



most probable theory being that it is a manifestation of degeneration. 

 Koch, 1 however, observed similar phenomena in the stomach of ticks 

 which had been previously infected with B. bigemina. 



Citrate of soda cultures have a special interest in connection with 

 Leishmania donovani, the parasite of kala-azar. By the addition of 

 an equal quantity of 2'5 to 5 per cent, citrate of soda solution to the 

 infected blood, the organisms not only remain alive for about four 

 weeks, but they reproduce themselves and pass into a developmental 

 stage which has, up to now, never been observed in man. At this stage 

 the organism is long and narrow in shape ; it possesses a flagellum, and 

 it is actively motile. In blood taken at the height of infection, shortly 

 before natural death, from an unchloroformed dog, active flagellate 

 forms of B. canis were found. The flagella of these organisms are, 

 however, so easily detached, that it is rarely possible to observe them 

 in fixed and coloured specimens. It has been suggested that these 

 flagellate forms of Babesia are the male sexual forms, but the 

 conclusion is very doubtful. 



Permanent pure cultures outside the body of the host have suc- 

 ceeded in the case of certain blood parasites, but upon two media 

 only. These are as follows : 



(1) Blood-agar (Novy) is used for the cultivation of Trypanosomes 

 and will be described later. It is equally useful for certain blood 

 parasites which have become adapted to cell-parasitism. These are 

 Hcemoproteus (Hartmann) 2 and Leishmania (Nicolle). 3 



(2) Blood-bouillon (Miyajima 4 ) yielded remarkable results with a 

 Babesia found in cattle in Japan and Corea. The infected blood was 

 withdrawn from the jugular vein and immediately defibrinized under 

 antiseptic precautions. It was then mixed with ordinary nutrient 

 bouillon in a proportion of 1 : 5 to 1 : 10 and kept in sterilized test-tubes 

 at a temperature of 20 to 30 C. Large round parasites, with a 

 diameter greatly in excess of that of the blood corpuscles, developed 

 in the fluid. (These appear to resemble forms found by Koch in East 

 Africa in the stomach of ticks which had been removed two to three 

 days previously, gorged with blood, from an ox infected with 

 B. bigemina). Flagellate forms are observed in old cultures after 

 seventy-two hours ; these increase in number and are most plentiful 



1 R. Koch, " Beitrage zur Entwickelungsgeschichte der Piroplasmen," Zeitsclir- 

 f. Hygiene, vol. liv, 1906, pp. 1-10. 



F. K. Kleine, " Kultivierungsversuch der Hundepiroplasmen," ibid., pp. 10-16. 



2 K. Kisskalt and M. Hartmann, " Praktikum der Bacteriologie und Proto- 

 zoologie." Jena : G-. Fisher, 1907. 



3 See F. Verdier, " Les Leishmanioses." Paris, 1908, p. 89, with plates. 



4 M. Miyajima, "On the Cultivation of a Bovine Piroplasma," Philippine Journal 

 of Science, vol. ii, 1907, pp. 83-91. 



