Usually, the reason for establishing xenic cultures will be for identification of the species of Entamoeba present in the sample, for example by isoenzyme analysis. Several intestinal species of Entamoeba can be found in humans which are sufficiently similar to cause diagnostic confusion and all can grow in the same media. Because E. histolytica is the only species that causes invasive disease, differentiation from the closely related, more common, and morphologically identical species E. dispar in particular is desirable, in order to prevent unnecessary chemoprophylaxis. Until less laborious methods become widely tested and implemented, isoenzyme analysis will remain the standard for separation of these two parasites.
The most common source of material will be stool samples and this is what is assumed below. In rare instances rectal biopsies or liver abscess aspirates have been the starting point for cultures. In the latter case, since the abscess is sterile, addition of a bacterial flora is necessary before inoculation of amebae into xenic culture. Such material has also been used for the direct establishment of monoxenic cultures. Unless the stool sample is from a patient with dysentery the amebae will be in the encysted form. This allows for several approaches to the establishment of cultures.
Elimination of unwanted organisms
One of the banes of xenic cultivation is the likelihood of unwanted organisms outgrowing the desired ameba. The most frequent source of this problem is Blastocystis hominis which may be the most common parasitic infection of humans. This organism is often missed on stool examination but grows luxuriantly in all the media used to cultivate xenic Entamoeba. Some authors control the growth of B. hominis with acriflavin but this also has an adverse effect on the bacterial flora and, directly or indirectly, on the ameba of interest. We have successfully used two methods to eliminate B. hominis from Entamoeba cultures.
The first method was described by Dobell and Laidlaw in 1926 (13). In this method, cysts are treated with 0.1N hydrochloric acid at room temperature for 10 minutes, washed thoroughly with distilled water, and re-inoculated into culture medium to which a suitable bacterial flora has been added. The acid kills the bacteria, any fungi, B. hominis, intestinal trichomonads, and any non-encysted amebae while leaving the cysts intact and viable. We have found that it is not necessary in most cases for the cysts to be mature. We do not know whether the cysts complete their maturation upon inoculation or whether immature cysts respond to the stimulus and excyst directly. The cysts used can be either from stool or from cultures - Entamoeba cultures in LE medium in particular frequently produce small numbers of cysts spontaneously.
The bacterial flora used in the above method is separated from another xenic culture by inoculating into culture medium, without rice starch, a small amount of supernatant from an established culture, subculturing twice and refrigerating the flora for 48 hours. The successful separation of the flora can be checked by inoculating a substantial volume into fresh medium with rice starch and checking for amebal growth. The flora can be stored at 4° C indefinitely.
The second method is that of Smedley (19) and is used when B. hominis appears in cultures. It does not rely on cysts being present and so has advantages over the method of Dobell and Laidlaw in that respect. However, the method may need to be repeated a couple of times before the B. hominis is completely eliminated. Cultures are pelleted and the pellet, which contains a mixture of all the organisms present, is resuspended in distilled water at room temperature for 15 minutes. The material is then re-pelleted and inoculated into fresh culture medium. Perhaps surprisingly, many Entamoeba trophozoites survive this treatment while B. hominis generally does not. A few cells of B. hominis may survive and start to grow, and the procedure will then need to be repeated. The advantage of Smedley's method is its simplicity 