886 PROTOZOOLOGY 



In solution A, Trichomonas sp. and Tricercomitus termopsidis were 

 cultivated. For Trichomonas termopsidis, a small amount of Loeffler's 

 blood serum and cellulose were added. All three flagellates were cul- 

 tured for over three years. In solution U to which 0.01 per cent blood 

 serum, cellulose and charcoal, were added, Trichonympha sphaerica 

 (from Termopsis angusticollis) grew well and multiplied up to two 

 weeks, although T. campanula and T. collaris failed to do so. The 

 culture in a test tube was inoculated with the entire hindgut of a 

 termite and kept at room temperature. 



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

 egg-white and 100 cc. of sterile Ringer's solution, to which a small 

 amount of yeast cake is added, is an excellent culture medium. Incu- 

 bation at room temperature; subcultures every 4-6 days. 



Trypanosoma and Leishmania. — Novy, MacNeal and Nicolle 

 (NNN) medium: 14 gm. of agar and 6 gm. of NaCl are dissolved by 

 heating in 900 cc. of distilled water. When the mixture cools to about 

 50°C, 50-100 cc. of sterile defibrinated rabbit blood is gently added 

 and carefully mixed so as to prevent the formation of bubbles. The 

 blood agar is now distributed among sterile test tubes to the height 

 of about 3 cm., and the tubes are left slanted until the medium be- 

 comes solid. The tubes are then incubated at 37°C. for 24 hours to 

 determine sterility and further to hasten the formation of conden- 

 sation water (pH 7.6). Sterile blood or splenic puncture containing 

 Trypanosoma cruzi or Leishmania is introduced by a sterile pipette 

 to the condensation water in which organisms multiply. Incubation 

 at 37°C. for trypanosomes and at 20-24°C. for Leishmania. 



For cultivating T. gambiense and T. rhodesiense, Tobie, von Brand 

 and Mehlman (1950) used the following medium: 



(a) Base. 1.5 gm. Bacto-beef, 2.5 gm. Bacto-peptone, 4 gm. sodium 

 chloride and 7.5 gm. Bacto-agar, are dissolved in 500 cc. distilled 

 water. After adjusting pH to 7.2-7.4 with NaOH, autoclave at 15 

 lbs pressure for 20 minutes. Cool this to about 45°C, then add whole 

 rabbit blood which had been inactivated at 56°C. for 30 minutes, in 

 the proportion of 25 cc. blood to 75 cc. base, using 0.5 per cent sterile 

 sodium citrate to prevent the coagulation. This base is placed in 

 test tubes (5 cc. each and slanted) or in flasks (25 cc), and allowed 

 to solidify. 



(b) Liquid phase. Sterile Locke's solution. This is added in 

 amounts of 2 cc. (to test tubes) or 10-15 cc. (to flasks), and cotton 

 plugs are applied. The trypanosomes are said to grow well and to 

 reach the peak population in 10-14 days. 



Entamoeba barreti. — Barret and Smith (1924) used a mixture of 



