178 RESEARCH IN PROTOZOOLOGY 



It should also be remembered that the amoebae never occur in 

 the cultures in numbers comparable to bacteria in cultures and 

 many fields of the microscope will usually have to be searched 

 before a single amoeba is encountered. This fact has caused inex- 

 perienced vs^orkers to regard cultures as negative w^hich were 

 actually positive and it should be the practice to examine at 

 least three preparations from a culture before returning a negative 

 report. In rich cultures the amoebae seldom number more than 

 two to four in one field of the microscope and many microscopic 

 fields will not show any amoebae. In cultures made directly from 

 the feces it is rare to observe even two amoebae in a microscopic 

 field but in transfers the amoebae are more numerous. 



If the technique recommended be employed it will be found 

 that cultures of the feces not infrequently give positive results 

 in individuals in whom the direct microscopical examination was 

 negative, and this is especially true if the feces are formed. 



In order to maintain strains of E. histolytica in culture it is 

 best to make subcultures every forty-eight hours and the modified 

 Locke-Egg-Serum culture medium is perhaps most satisfactory for 

 this purpose. Certain precautions are necessary in order to prevent 

 the loss of the culture, the most important being the maintenance 

 of the cultures at a temperature between 37° and 38° C, the 

 prevention of bacterial contamination other than that originally 

 present in the culture, and the use of a carefully made medium 

 in which no variation occurs in the quantity of the constituents. 

 It is especially important that the proper temperature be main- 

 tained, for this parasite is very sensitive to changes in temperature, 

 especially to cold, and we have lost many strains by reason of the 

 electric current of our incubators failing at times. It is also 

 necessary to use aseptic precautions in making subcultures, for a 

 change in the bacterial content of the cultures frequently results 

 in the death of the amoebae. Having obtained a strain of E. his- 

 tolytica in culture it is best to prevent bacterial contamination with 

 bacteria not present in the original culture, for the introduction 

 of other bacteria frequently leads to the death of the amoebae. In 

 our laboratories the introduction of such foreign bacteria, through 

 failure of asepsis in transferring cultures, has frequently led to 

 the loss of the strain of the parasite under cultivation. 



Care should also be taken to transfer a considerable amount 

 of the sediment in the cultures as often the amoebae are present 



