Differentiation of Entamoeba: Encystation of E. invadens in Monoxenic and Axenic Cultures

Abstract

A method to stimulate mass encystation of Entamoeba invadens in monoxenic or axenic cultures, using a simple monophasic medium, has been developed. The procedure involves transfer of trophozoites from an axenic growth medium (TPS-1) to an encystation medium containing serum which has been conditioned by growth of bacteria, either prior (axenic) or subsequent (monoxenic) to inoculation with amebae. In encystation media lacking conditioned serum trophozoites do not encyst and generally lyse. Several strains of bacteria can effectively condition the encystation medium; best results were obtained using Proteus morganii. Axenically produced cysts are indistinguishable from monoxenic cysts and excysted in growth medium. Mass encystation of the reptilian parasite, Entamoeba invadens, can be stimulated in cultures of amebae growing with bacteria in media containing insoluble starch (Balamuth, 1962); however, the presence of a metabolically active second organism and insoluble components makes this an undesirable system with which to study the mechanism of differentiation by these parasites and complicates the isolation of pure cysts. We have developed a method whereby mass encystation of E. invadens can be consistently induced in either monoxenic or axenic cultures using a simple monophasic medium. The procedure involves transfer of trophozoites from a rich axenic growth medium to a simpler encystation medium which is conditioned by growth of bacteria, either prior (axenic) or subsequent (monoxenic) to inoculation with amebae. This method facilitates preparation and isolation of E. invadens cysts and has provided a system with which to analyze environmental and metabolic factors controlling differentiation (Bailey et al., 1970). Encystation results using this technique and the consequences of varying several parameters are described here. MATERIALS AND METHODS Ameba growth and encystation Trophozoites of Entamoeba invadens (strain IP-1) were grown axenically at approximately 25 C in Diamond's TPS-1 medium (Diamond, 1968) using techniques similar to those described by him for axenic culture of Entamoeba histolytica. Received for publication 8 April 1971. * To whom all correspondence should be addressed: c/o The Rockefeller Foundation, Box 2453, Bangkok, Thailand. With inocula of 1 X 104 to 3 X 104 cells per ml, cell numbers increased approximately 10-fold in this medium, and multiplication ceased after 10 to 14 days. Cysts sometimes formed in growth cultures but their number never exceeded about 10% of the total number of amebae. The encystation medium was derived from one used by Morgan et al. (1968). It contained, per liter, 4 g NaCl, 2 g Difco yeast extract, 1.6 g KH2P04, and 3.2 g K2HPO4. This was adjusted to pH 7.0 and after autoclaving was diluted with onehalf volume of filter-sterilized horse or calf serum. For monoxenic encystation, this medium was inoculated simultaneously with amebae and bacteria. For axenic cultures, the desired volume of encystation medium, which varied from 20 to 500 ml depending on experimental needs, was first inoculated with a loop of bacteria from stock slants and incubated for 24 hr at 37 C in filled, sealed, tubes or flasks. In all cases cultures reached late stationary phase in this time. Bacterial growth during conditioning was followed by the change in absorbency of the culture at 600 nm. The bulk of the bacteria were then removed by centrifugation and the conditioned encystation medium was sterilized by filtration through a type GS Millipore filter prior to inoculation with amebae. Axenic conditioned encystation medium remained active indefinitely when stored at -20 C. Ameba trophozoites were transferred to encystation media in the following way: growth cultures were chilled at 5 C for 5 min to release trophozoites from the vessel wall and break clumps of amebae. The protozoa were suspended evenly and replicate samples removed and counted in hemocytometer chambers. The amebae were then centrifuged for 5 min at 500 g, and the supernatant medium decanted. Unconditioned encystation medium (complete or minus serum) was added to resuspend the trophozoites at the desired concentration. Volumes of 0.05 to 0.1 ml of this suspension were then pipetted into each ml of the appropriate encystation medium. Encystation cultures were contained in filled, 9-ml screw-capped tubes and incubated horizontally at 25 C. Sterility of axenic cultures was checked at the time of inoculation and