1.A method for obtaining cysts of E. histolytica free of living bacteria is described.
2.These bacteria-free cysts usually excyst when suspended in various media with living bacteria. Excystation of the bacteria-free cysts in the absence of bacteria was observed only in the presence of the reducing agents, cysteine or neutralized thioglycollic acid, or under conditions of reduced oxygen tension produced by aeration with nitrogen gas or by alkaline pyrogallol. With all of these methods, excystation was correlated with a decrease of the oxygen tension of the medium as indicated by the reduction of methylene blue.
3.Excystation of bacteria-free cysts of E. histolytica was observed in a medium containing only inorganic salts (modified Locke's solution).
4.Anaerobic cultures of motile forms of E. histolytica with bacteria showed a greater sensitivity to oxygen than did aerobic cultures.
5.Continued cultivation of the bacteria-free excysted forms has not yet been effected without the addition of living bacteria.
6.On the basis of these observations it was concluded that:
(a)In addition to water and certain salts, a factor usually supplied by living bacteria is required for excystation of bacteria-free E. histolytica cysts. This factor is probably any agent that reduces the oxygen tension of the medium.
(b)Other factors in addition to the above are required for continued cultivation and “normal multiplication” of bacteria-free excysted forms of E. histolytica. These factors are not furnished by heat-killed bacteria. They have not yet been discovered, although a number of nutrient media have been tested.