ENTAMCEBA HISTOLYTICA 207 



and sterilized by steaming for twenty minutes on three successive days. 

 As in the case of the egg medium, a few drops of blood are added before 

 inoculation. 



Attempts were made to cultivate amoebae from forty-five samples of 

 fseces containing E. histolytica, and amoebas were grown from six which 

 contained blood and mucus. Bacteria grew in the media as well as the 

 amoebae, and it was necessary to subculture every twenty-four to seventy- 

 two hours on account of the quantity of acid produced by the bacteria. 

 A temperature of 28° to 30° C. was better than a higher one, as bacterial 

 growth was reduced. Subculture was effected by transfer of 0-5 to 1 c.c. 

 of the culture. By this means cultures were maintained for over three 

 months, and not only did multiplication of the amoebae take place, but 

 encystment also occurred. Cats were infected by inoculation per rectum 

 with cultures of more than two and a half months' standing, and typical 

 dysenteric symptoms with amoebae resulted, while post-mortem examina- 

 tion showed the characteristic amoebic lesions, from which fresh culture 

 was obtained. Other animals were infected by feeding them on cultures 

 containing cysts. Dobell (1919) stated that he attempted without 

 success to cultivate E. histolytica by this method, and concluded that there 

 must have been some fallacy in Cutler's work. The writer also failed to 

 repeat Cutler's experiments. Barret and Smith (1923, 1924), however, 

 obtained cultures of another amoeba, Entamoeba barreti of the turtle, 

 Chelydra serpentina. The medium used was a mixture of human blood- 

 serum 1 part and 0-5 per cent, sodium chloride solution 9 parts. In 

 each tube 5 c.c. of the mixture was used. A small quantity of mucus 

 obtained from the intestinal wall was inoculated at the bottom of the 

 tubes, which were kept at 10° to 15° C, or at room temperature. At first 

 it was necessary to subculture every twenty-four or forty-eight hours, 

 but when a culture was established a weekly transfer was sufficient. Two 

 strains were kept for nine months, during which thirty subcultures were 

 made. The amoeba? multiplied actively, and corresponded in every way 

 with those seen in the intestine of the turtles. No cysts were found, 

 however. Cultures of E. ranarum of the frog have also been obtained. 

 These results, which were obtained with amoebae of cold-blooded hosts, 

 led Barret and Smith to suggest that Cutler may have been more successful 

 with E. histolytica than some had supposed. 



Quite recently Boeck and Drbohlav (1925) have cultivated E. histolytica 

 on solid egg and blood agar slopes covered with Locke's solution containing 

 serum or egg albumin. From two human cases E. histolytica was isolated 

 and maintained in subculture for many generations, in one case for more 

 than eight months, during which 150 subcultures were made. Sub- 

 culture was made every two or three days, and the tubes were kept at 



