INTKODUCTION. 39 



These cultures are incubated at 37° to 40° C, and the forms 

 which appear are those which normally occur in the Vertebrate 

 host. Bass and Johns (1912) were the first to succeed in 

 cultivating malarial parasites. Their methods, with shght 

 modifications, were successfully followed by Ziemann (1913, 

 1914), J. G. & D. Thomson, (1913), Row (1917), Suiton (1922), 

 and others. Knuth and Richters (1913), Ziemann (1913, 

 1914), and Thomson and Fantham (1914) were the first to 

 apply these methods to the Piroplasmids. The technique of 

 some of these methods is given below : — 



Bass and Johns's Method. — Withdraw 10 c.c. of malarial blood 

 from a vein with a syringe, and transfer immediately to a de- 

 fribinating flask containing 0-1 c.c. of 50 per cent, solution of 

 dextrose. Defibrinate by stirring with a glass rod. Transfer 

 the defibrinated dextrose blood to culture-tubes not less than 

 1-25 cm. in diameter and 12-5 cm. in depth. The quantity 

 of blood in each tube may vary in depth from 2-5 to 5 cm., 

 which will give a column of serum 1-25 to 2-5 cm. deep above 

 the cells, when they have settled to the bottom of the tube. 

 Incubate the tubes in the vertical position at a temperature 

 of 40° C. The parasites live and develop in the red blood- 

 corpuscles just below the surface of the deposit. Carefully 

 withdraw the cells from this layer by means of a fine pipette 

 and examine at intervals. The young trophozoites will be seen 

 to grow into schizonts and break up into merozoites. Some 

 of these may enter other corpuscles and grow into schizonts, 

 but as a rule those escaping from the corpuscles are devoured 

 by the leucocytes. To remove the leucocytes centrifuge 

 the defibrinated glucose blood at a speed sufficient to cause the 

 leucocytes to occupy the upper layer of the deposit. Transfer 

 the supernatant serum to flat-bottomed culture-tubes, fllhng 

 to a depth of 1-25 to 2-5 cm. Pass a pipette into the middle 

 of the deposit in the centrifuge -tube, draw off the red corpuscles 

 with the parasites, and transfer them to the bottom of the 

 culture -tubes. In this way, in the absence of the leucocytes, 

 the parasites may complete two or three cycles ; but it has not 

 been found possible to cultivate them for more than three 

 generations. For making subcultures, take normal blood, 

 treat it in the same manner, and inoculate it by means of 

 a pipette with infected blood from the previous culture. 



Row (1917) and Sinton (1922) have devised modifications 

 by which the growth of a single generation can be followed with 

 a few drops of blood. 



Row's Method. — Draw the blood from the finger into a smaU 

 tube and defibrinate it in the same. Transfer the small 

 quantity of defibrinated blood by means of a pipette to a small, 

 flat-bottomed tube containing serum to which the requisite 

 quantity of dextrose has been added. Place the small tube 



