, PROTOZOA 139 



a quantity of chopped hay in water is an easy dnd valuable method of preparing 

 culture media. For the cultivation of amoebae, the following media is widely em- 

 ployed. It should be noted, however, that the amoebae which have been cultivated 

 are regarded as free-living forms and the attempts to cultivate parasitic amoeb* 

 have thus far been unsuccessful. 



Medidm of Musgrave and Clkgg 



"^Sar 20 to 30 g. 



Liebig's extract of beef 3 to .5 g. 



Common salt 3 to . s g. 



Water i^ooo c.c. 



This medium is designed to provide for slow bacterial growth in order to provide 

 food for amoebae. On a richer medium the latter are overwhelmed by the rapid 

 growth of bacteria. 



For the cultivation of trypanosomes, leishmania and other flagellates the so- 

 called triple N media is employed. This is prepared as follows: 



NicoLLE, NovY, MacNeal Medium 



Water poo c.c. 



Salt 6 g. 



Agar 16 g. 



Dissolve, distribute in tubes, sterilize and add to the medium in each tube after 

 Uquefying and cooling to 40°-5o°C. one-third its volume of rabbit blood obtained by 

 cardiac puncture. Slope the tubes for twelve hours, incubate at 37°. for five days 

 to test the sterility of the medium and then keep them at the ordinary temperature 

 of the laboratory for a few days before sowing them. (The tubes should be sealed to 

 prevent evaporation.) 



The malaria organisms have been made to continue development outside the body 

 by the following method devised by Bass. 



Bass's Method. — The blood in 10- to 20-c.c. quantities is taken from the patient's 

 vein and received in a centrifuge tube which contains }{o c.c. of 50 per cent, glucose 

 solution. A glass rod, or piece of tubing, extending to the bottom of the 'centrifuge 

 tube is used to defibrinate the blood. After centrifugalizing there should be at least 

 I inch of serum above the cell sediment. The parasites develop in the upper cell 

 layer about J^o to J^o iiicli from the top. All of the parasites contained in deeper 

 lying red cells die. To observe the development, red cells from this upper J^o-inch 

 portion are drawn up with a capillary bulb pipette. 



Should the cultivation of more than one generation be desired, the leucocyte 

 upper layer must be carefully pipetted off, as the leucocytes immediately destroy the 

 merozoites. Only the parasites within red cells escape phagocytosis. Sexual 

 parasites are much more resistant, and the authors think they observed partheno- 

 genesis. The temperature should be from 40 to 41°. and strict anaerobic conditions 

 observed. iEstivo-autumnal organisms are more resistant than benign tertian ones. 

 Dextrose seems to be an essential for the development of the parasites. 



