716 PROTOZOOLOGY 



dium: 5 cc. of Bacto nutrient agar dissolved in boiling water, is 

 tubed and the tubes are autoclaved for 15 minutes at 15 pounds 

 pressure and slanted. After cooling, a modified Ringer's solution is 

 added to the agar slants to a height of 2-3 inches. The composition 

 of Ringer's solution used, is as follows : 



Sodium chloride 6 gm. 



Potassium chloride . 1 gm. 



Sodium bicarbonate . 1 gm. 



Calcium chloride 0.1 gm. 



Glass distilled water 1000 cc. 



Loeffler's dried blood serum 2 . 5 gm. 



The tubes are inoculated with vaginal material and incubated at 

 36-37°C. Transfers are made every 3 or 4 days. 



Lophomonas hlattarum 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. 



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

 9 parts of 0.5% NaCl and 1 part of human blood serum. Incubation 

 at 10-15°C. 



E. invadens. — Ratcliffe and Geiman (1938) used a mixture of 

 gastric mucin 0.3 gm., "ground alum" salt 0.5 gm., and distilled 

 water 100 cc. About 2 mg. of sterile rice starch is added to each cul- 

 ture tube at the time of inoculation. Culture at 20-30° C. and sub- 

 culture every 7 days. 



E. histolytica and other amoebae of man. — The first successful cul- 



