718 PROTOZOOLOGY 



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. 



Plasmodium.— BsLSS and John's (1912) culture is as follows: 10 cc. 

 of defibrinated human blood containing Plasmodium and 0.1 cc. of 

 50% sterile dextrose solution are mixed in test tubes and incubated 

 at 37-39°C. In the culture, the organisms develop in the upper layer 

 of erythrocytes. Although several attempts have since been made 

 by several workers, Plasmodium has not been cultivated in vitro for 

 more than a few generations. 



Balantidium coli. — Barret and Yarb rough (1921) first cultivated 

 this ciliate in a medium consisting of 16 parts of 0.5% NaCl and 1 

 part of inactivated human blood serum. The medium is tubed. 

 Inoculation of a small amount of the faecal matter containing the 

 trophozoites is made into the bottom of the tubes. Incubation at 

 37°C. Maximum development is reached in 48-72 hours. Subcul- 

 tures are made every second day. Reese used a mixture of 16 parts 

 of Ringer's solution and 1 part of Loeffler's dehydrated blood serum. 



Atchley (1935) employed a medium composed of 4 parts of Ringer's 

 solution and 1 part of faeces, which is filtered after 24 hours, centri- 

 fuged and sterilized by passage through a Seitz filter. Nelson (1940) 

 also used 1 part of caecal contents of pig in 9 parts of Ringer's solu- 



