410 FAMILY: TRYPANOSOMID^ 



McNeal's rabbit blood-agar, and in a simplified medium now known as 

 the N.N.N. (Novy, McNeal, Nicolle) medium. Furthermore, in this 

 medium subculture was readily obtained, so that the flagellate could be 

 maintained as easily as any bacterial organism. Since that time the 

 culture method has been universally adopted, and is a recognized aid to 

 diagnosis. A strain of L. donovani isolated in 1910 was reported by 

 Nicolle (1925) to be still growing in N.N.N, medium. During these years 

 it has been subcultured 395 times. 



The change undergone by the leishmania when introduced into the 

 medium can be studied in heavy infections by examination of the inocu- 

 lated material at frequent intervals. According to Leishman and Statham 

 (1905), who studied the development, the first change is an increase in 

 size of the leishmania, a growth of the nucleus, and an increased vacuo- 

 lization of the cytoplasm. In many cases the leishmania become pyriform 

 in shape. After forty-eight hours, growth of the flagellum commences. 

 It is a rapid process, and takes place from an eosin staining vacuole or 

 body which lies in front of the kinetoplast. This body moves to that end 

 of the organism which will be its anterior end in subsequent development. 

 Its contents are extruded as a series of fine filaments, which unite and 

 form the rudiments of the flagellum. As this process was studied only 

 in dried films, it is probable that the appearances are artefacts due to a 

 rupture of the vacuole. It is far more probable — and this is supported 

 by what is known of the formation of flagella in other organisms — that 

 the axoneme, which is sometimes visible in the leishmania, continues its 

 growth, and extends through the surface of the parasite to form the 

 flagellum. Active flagellate forms may be seen in cultures at any time 

 between forty-eight and seventy-two hours after the medium has been 

 inoculated. Very soon many of the organisms become still more elon- 

 gated till the body measures from 10 to 20 microns in length, while the 

 flagellum is often longer than the body. The fully-formed flagellate is 

 flattened like a blade of grass. Sometimes one edge of the organism is 

 convex and the other slightly concave, giving it the shape of a curved 

 sword-blade. In a culture of four or five days' growth various types of 

 flagellates are present, including round forms 4 to 5 microns in diameter 

 with long flagella, broad pyriform individuals with rounded anterior and 

 tapering posterior ends, and measuring about 10 microns in length and 

 4 to 5 microns in breadth, and the longer sickle-shaped forms already 

 mentioned (Fig. 190). The various types are connected by intermediate 

 forms. Reproduction by longitudinal division takes place rapidly till the 

 culture at the end of a week to ten days may be swarming with flagellates. 

 At division the blepharoplast divides, and a new axoneme is formed by 

 outgrowth from the blepharoplast. The parabasal then becomes constricted 



