Mitosis and polykaryon formation of Entamoeba histolytica in axenic cultures

Axenic cultures of E. histolytica were established in Coplin jars containing glass slides. The cultures were harvested after 72 hours, fixed and stained with Giemsa and acridine orange. Sedimented parasites were used for inclusion in methacrylate for thin sections that were stained with acridine orange, and epon-araldite for ultrathin sections for electron microscope study. The Giemsa stain showed trophozoites of different sizes and variation in the shape, size and number of the nuclei. Division occurred by budding, sometimes by fission, with occasionally apparent extrusion of nuclear material into the cytoplasm. Acridine orange demonstrated DNA in the center of the nuclei and within intranuclear small vesicles. The electron microscope showed mono and multinucleated amoebae with irregular nuclear membranes, coarse material attached to the nuclear membrane, and sometimes intranuclear parallel microtubules without nuclear attachment. There were structures around the karyosome in some nuclei resembling chromosomic material of superior eukaryotes, without kinetochore, as well as intranuclear small vesicles containing sausage-shaped structures. The findings suggest that E. histolytica undergoes polyploidy evidenced by giant nuclei, followed by segregation of chromatin in smaller nuclei, by budding and production of polykaryons of different nuclear size. Multiple small intranuclear vesicles containing structures that may represent forms of rRNA thus contributing to ribosomes. Absence of cytokinesis is the cause of the production of polykaryons with an asynchronous rate of nuclear and cytoplasmic division. Polynucleation also could be related in the origin of cyst formation in amoeba, as unknown selective forces may induce amoebae the synthesis of chitin capsule.