888 PROTOZOOLOGY 



NaCl 9 gm. 



KC1 0.2 gm. 



CaCl 2 0.2 gm. 



Distilled water 1000 cc. 



The covering liquid is serum-Ringer or egg-albumin. The latter is 

 prepared by breaking one egg white in 250 cc. of Ringer's solution 

 which is passed through a Seitz filter. Before inoculating with amoe- 

 bae, a small amount of sterile solid rice-starch (dry-heated at 180°C. 

 for 1 hour) is added to the culture tube. 



(d) Horse-serum-serum (HSS) or Horse-serum-egg-albumin (HSA) 

 medium. Whole horse-serum, sterilized by filtration, is tubed and 

 slanted at 80°C. for about 60-70 minutes (do not heat longer). 

 When the slants have cooled, they are covered with diluted serum or 

 egg-albumin given for (c). The tubes are incubated for sterility and 

 sterile rice-starch is added immediately before inoculation. Frye and 

 Meleny (1939) substituted the liquid portion of this medium by 

 0.5% solution of Lily liver extract No. 343 in 0.85% NaCl. 



(e) Liver-agar-serum (LAS) medium. Cleveland and Sanders 

 (1930) used the following medium: 



Liver infusion agar 



(Difco dehydrated) 30 gm. 



Glass distilled water 1000 cc. 



The medium is tubed, autoclaved, and slanted. The slants are cov- 

 ered with a 1:6 dilution of sterile fresh horse serum in 0.85% NaCl 

 solution. A 5 mm. loop of sterile rice flour or powdered unpolished 

 rice is added to each tube. In making subculture, remove 2 or 3 drops 

 of the rice flour debris from the bottom with a sterile pipette. 



(/) Egg-yolk-saline medium (Balamuth and Sandza, 1944). Two 

 eggs are hard-boiled. Upon cooling, the egg white is discarded and 

 the yolks are crumbled in a beaker containing 125 ml of 0.8 per cent 

 sodium chloride solution. The mixture is boiled for 10 minutes, and 

 after replacement of evaporated water the infusion is filtered by 

 suction pump and restored to 125 ml. The filtrate is autoclaved 20 

 minutes at 15 pounds pressure. Upon cooling, a slight precipitation 

 of yolk settles, and is removed by simple filtration, after which 

 125 ml of N/15 phosphate buffer (pH 7.5) is added, making the 

 total salt concentration N/30 phosphate solution in 0.4 per cent 

 sodium chloride. This final mixture is tubed in 5 ml amounts, auto- 

 claved as before, and then is stored under refrigeration until use. 

 Before introducing amoebae a loop of sterile rice starch is added to 

 each tube. 



