174 RESEARCH IN PROTOZOOLOGY 



solution contains 9 grams of sodium chloride, 0.2 gram of calcium chloride, 

 and 0.2 gram potassium chloride to the litre of distilled water. The covering 

 fluid for the egg slant is egg albumin or inactivated horse serum diluted 

 one part to eight with the Ringer solution. Just before making the culture 

 for amoebae a small amount of rice starch which has been sterilized is added 

 to the medium under aseptic precautions. The rice starch is sterilized by 

 heating in a dry air oven at 180° C. for one hour, about 2.5 grams of the 

 starch being placed in a small glass tube. If egg albumin is used instead 

 of horse serum the whites of four eggs are mixed with a litre of Ringer 

 solution and the whole sterilized by filtration. No adjustment of the reaction 

 of this medium is necessary. 



Medium No. 2. This medium is similar to Medium No. i except that un- 

 diluted horse serum is used for the slants instead of egg and Ringer solution. 

 The horse serum is sterilized by filtration, tubed, slanted and inspissated at 

 80° C. for one hour or an hour and ten minutes and no longer. The tubes 

 are then cooled, incubated for sterility, and then the slant covered with egg 

 albumen solution or serum prepared as in Medium No. i. Starch is added 

 at the time of culture as described above. No adjustment of the reaction of 

 this medium is necessary. 



Dobell claims that E. histolytica grows more abundantly and 

 lives longer upon these media, especially the horse-serum-egg- 

 albumen-starch medium, than upon the media of Boeck and 

 Drbohlav, sometimes living for as long as two weeks and that sub- 

 culture is not necessary more often than once a week. The starch 

 also prevents the development of Blastocystis Jwininis and re- 

 places the dextrose used in the Boeck and Drbohlav media. 



Craig's Media. In the writer's observations upon the cultivation 

 oi E. histolytica, the solid ^gg or serum slants used in the media 

 just mentioned have not been found to be necessary and the 

 belief of Kofoid and Wagener (1925) that either the coagulated 

 Qgg or blood contained in blood agar slants is required for at least 

 a part of the food supply of this amoeba, has been found to be 

 erroneous. In 1926, the writer published a simplified method for 

 the cultivation of this parasite in which no solid substratum 

 was used, and in a later paper (1926a) published still more simple 

 methods for cultivating the organism. The observations noted 

 demonstrated that none of the constituents of either Locke or 

 Ringer's solution, except possibly the sodium chloride content, 

 are absolutely essential for the cultivation of E. Jiistolytica, as a 

 simple mixture of normal salt solution and inactivated human blood 

 serum is a suitable culture medium for this parasite, although it 

 cannot be maintained in this simple medium indefinitely, as it 



