416 HANDBOOK OF PROTOZOOLOGY 



of the filtrates in each test tube. Autoclave the test tubes for 

 15 minutes under 15 pounds pressure. After cooling, a small 

 amount of fresh fecal matter containing the flagellates is intro- 

 duced into the tubes. Incubation at 37° C. 



Lophomonas blattarum and L. striata. — A mixture of one 

 part sterile egg albumen and three parts autoclaved Ringer's 

 solution, to which a small amount of yeast cake has been added, 

 is excellent for the culture of these cockroach flagellates. In- 

 cubation is at room temperature, and subcultures are made 

 every fourth day. 



Trypanosoma and Leishmania. — -Blood-agar medium, com- 

 monly called N.N.N, medium because it was originally de- 

 veloped by Novy and MacNeal and simplified by Nicolle, is 

 prepared in the following manner: 14 gm. of agar and 6 gm. 

 of sodium chloride are dissolved by heating in 900 c.c. of dis- 

 tilled water. When the mixture cools to about 50° C, 50 to 

 100 c.c. of sterile defibrinated rabbit blood is gently added and 

 carefully mixed so as to prevent the formation of bubbles. 

 The blood agar is now quickly distributed among sterile test 

 tubes to the height of about 3 cm., and the test tubes are left 

 in a sloping position until the medium becomes solid. The 

 tubes are then incubated at 37° C. for 24 hours to determine 

 sterility and further to hasten the formation of condensation 

 water. Sterile blood containing Trypanosoma or Leishmania 

 is introduced by a Pasteur pipette to the condensation water, 

 in which the organisms multiply. This medium is also good for 

 intestinal flagellates and amoebae of vertebrates. 



Entamoeba harreti.- — Barret and Smith used a mixture of 

 9 parts of 0.5 per cent sodium chloride and 1 part of human 

 blood serum. After inoculation the culture is incubated at 

 from 10 to 15° C. 



Entamoeba histolytica and other intestinal amoebae. — Since 

 Boeck and Drbohlav (1925) successfully cultivated Entamoeba 

 histolytica by using a medium composed of Locke's solution, 

 egg, and human blood serum, several authors have repeated 

 their experiments and have obtained positive results. This 

 method has been modified and simplified in various ways. Per- 

 haps the simplest of all is that proposed by Craig as salt-serum 

 (or S.S.) medium, in which 0,85 per cent sodium chloride solu- 



