Large Lot Cultivation of Entamoeba histolytica

The construction and use of a positive-pressure, anaerobic incubator for the cultivation of large lots of Entamoeba histolytica is described. Using commercially available prepurified nitrogen as the blanketing gas, harvests of 2 to 4.3 ml of clean, packed, active trophozoites were obtained from 1 liter of culture medium spread in thin layers in glass dishes and incubated for 48 to 72 hr at 36 to 37 C. The culture fluid contained trypticase, glucose, cysteine, thiomalate, and salts to which was added sterile horse serum, penicillin, and washed cells of an organism provisionally designated Bac- teroides symbiosus. The quantity of added bacterial cells appeared to be the growth-limiting factor since about 3 to 4 million trophozoites were produced per mg of added bacterial nitrogen. Nine strains of amebae were tested and all were found to grow satisfactorily in the anaerobic incubator. A total of 63.5 ml of packed trophozoites (14.8 billion cells) were harvested during about 6 months operation of an incubator. The fresh, frozen, or lyophilized trophozoites were suitable for biochemical or serological investigations.