APPENDIX ON PROTOZOOLOGY 743 



Mtisgrave and Clegg in 1904 devised a culture medium for 

 amoebae. The organisms grown by them were probably not dysen- 

 teric amoebae, as was thought, but free-living forms. Phillips 1 (1915) 

 gives a slightly modified formula of Musgrave and Clegg's medium, 

 thus : 



Agar-agar ,.. ... ... ... 2-5 grm. 



Sodium chloride ... ... ... ... 0-05 ,, 



Liebig's beef extract ... ... ... 0-05 ,, 



Normal sodium hydroxide ... ... ... 2*0 c.c. 



Distilled water ... .., ... ... ico'O ,, 



Without clarifying, sterilize at 7 kilograms pressure per square centi- 

 metre for about three-quarters of an hour. It should be neutral to 

 phenolphthalein. 



Anna W. Williams 8 (1911) described a medium consisting of fresh 

 tissue spread on agar plates for the culture of amoebae. There are 

 three stages in the procedure : (i) obtaining living amoebae free from 

 other living organisms ; (2) obtaining sterile tissue ; and (3) making 

 successive transplants of amoebae and tissue, and showing that every 

 transplant is free from other living organisms. Each step requires 

 many controls. The essentials of the method may now be given. 

 Remove aseptically and rapidly the tissue required, such as brain, 

 liver, kidney, or spleen, from a freshly killed animal (guinea-pig, 

 rabbit, or dog). Put each tissue on a separate agar plate. Cut the 

 selected tissue into tiny pieces, and spread them over freshly made 

 agar plates. Place these plates in a thermostat at 36 C. for twenty- 

 four hours to insure sterility. Add the broken up tissue to the 

 amoebae, free from bacteria, and maintain the cultures in thermostats, 

 some at 36 C., and some at 20 C. to 24 C. Emulsions of liver and 

 brain in sterile neutral glycerine may also be used. The freshly 

 removed tissue serves as food for the amoebae. 



The cultural amoebae mentioned on p. 42 were grown on such 

 media or modifications thereof. One modified medium actually used 

 was brain tissue, to which blood was added from day to day, and an 

 easily assimilable bacterium (one of the influenza group of bacilli) 

 was present, which did not overgrow the medium at a temperature of 

 38 C. Different conditions of food and of temperature produced 

 morphological variations in the cultural amoebae. 



Couret and J. Walker 3 (1913) state that they have cultivated five 

 varieties of intestinal amoebae, the associated bacteria having been 

 previously separated. They used a medium consisting of agar to 



1 " Amoebiasis and the Dysenteries," p. 8. 



Journ. Med. Research, xxv, p. 263 ; and Proc. Soc. Exper. Biol. and Med., viii, p. 56. 



3 Journ. Exper. Med., xviii, p. 252. 



