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SYNOPSIS. Germfree and conventional guinea pigs and germfree rats were inoculated with large numbers of axenically cultivated Entamoeba histolytica. Amebic lesions were not found in any of the animals, and there was no indication that the ameba had established lumen infections in any instance. The axenic amebae appeared to have lost their pathogenicity. This loss was believed to depend, in part at least, upon the fact that they did not encyst and thus did not complete their life cycle in axenic culture.REVIOUS investigations on the response of germfree animals P to intracecal inoculation with Entamoeba histolytica have employed inocula containing Trypanosoma cruzi culture forms along with the ameba ( 9 ) . The axenic in vitro cultivation of E. histolytica (2) has made it possible to obtain amebic inocula free from all other microorganisms. I t seemed of interest to ascertain if amebae adapted to axenic growth in vitro would grow axenically in vivo and produce lesions. The virulence of these amebae was evaluated further by introducing them intracecally into a strain of guinea pig susceptible to amebic enteritis. MATERIALS AND METHODSThree strains of E. histolytica were used: 200-NIH (12), HK-9 (4), and ABRM (isolated from a rectal abscess by Dr.B. Sepulveda and Miss M. de la Torre, Hospital General Centro Medical Nacional, Mexico City, Mexico). Axenic cultures of ameba grown in either Diamond's TTP-S diphasic, or TP-S-1 monophasic medium in Screw-cap tubes (3) served as the source of the parasites. The inocula were prepared from 72-hr cultures by chilling the tubes briefly in ice water to free amebae from walls of the tubes, centrifuging at 9OOg for 3 min, and pouring off the supernatant fluid. The remaining amebae were counted in a hemocytometer.Germfree animals were Hartley strain guinea pigs and Wistar strain rats. Guinea pigs were obtained by Caesarean section and maintained on the L-445 diet (11). Rats were obtained from the colonies of the Germfree Unit, Division of Research Services, NIH, and maintained on diet L-356 (5). All germfree animals were housed in series 500 stainless steel isolators (Reyniers Germfree System). The isolators were monitored microbiologically as in previous studies ( 11 ). Conventional guinea pigs were NIH strain obtained from the colonies at NIH and maintained on commercial guinea pig chow supplemented daily with fresh kale. These animals were housed in standard stainless steel cages in a conventional animal room. High susceptibility of this strain to induced amebic enteritis was determined previously (unpublished data).Fifty-five 18 day old germfree guinea pigs were used in the experiments. Amebae were injected into the cecum of laparotomized animals that were anesthetized with sodium pentothal. The majority of guinea pigs received 1.0 ml usually containing 3-5 x 105 amebae, but 4 animals were inoculated with 5 X 106 parasites. All animals were examined microscopically for enteric lumen amebae and also for gross enteric and other visceral amebic lesions at postinoculation interva...