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 and the fact that no separate bacterial flora is needed.
Other unwanted organisms such as fungi and trichomonads will usually disappear from xenic cultures after several passages. However, occasional instances of balanced mixed cultures are known.
In our experience LE has proven to be the best medium for primary isolation of Entamoeba species from stool, although we have limited experience with Robinson's medium which is widely used by others for this purpose. TYSGM-9 can also be used for isolation but its primary utility is in generating large numbers of amebae from established cultures. The numbers of amebae obtained from the two diphasic media are generally low in comparison with TYSGM-9, but their success in primary isolation amebae from microscopically positive stool is higher. In all cases, rice starch is added to the medium before inoculation, as are the antibiotics when needed.
Material for inoculation of xenic cultures can be prepared in several ways (9). Most commonly, stool samples are emulsified in saline and passed through a mesh to remove most of the larger particulates from the material before addition to the culture medium. However, small-pea sized pieces of fecal material can be added directly to the medium. It is always a good idea to include portions of the stool that appear mucoid or bloody if these are present. Stool fractionation by flotation in zinc sulfate is also used as this reduces the amount of debris while concentrating the cysts present in the sample. We routinely use more than one medium, if available, and set up duplicate cultures where one has antibiotics added and the other not. Penicillin/streptomycin or erythromycin are the antibiotics of choice as they appear to have little direct effect on the amebae. However, the widespread occurrence of antibiotic resistance in bacteria makes it impossible to generalize about the amount and type of antibiotics necessary to control the growth and rice-splitting activity of human bacterial flora.
Culture tubes, containing medium and rice starch, to which stool derived material has been added are incubated vertically at 35.5° C for 48 hours before examination. Ideally, examination should be done in situ if possible. This can be accomplished by slanting the tubes and using an inverted microscope. Amebae can be observed adhering to the walls of the glass culture tubes above the fecal material (and the slant in diphasic media). In situ examination is much easier in monophasic medium due to its relative clarity. Alternatively, a drop of sediment can be extracted from the tube for examination on a microscope slide. If no growth is observed at 48 hours a blind passage should be made. The culture tube is chilled in an ice-water bath for 5 minutes (to release amebae adhering to the glass) and most of the liquid overlay discarded to leave less than 1 ml in the tube. The sediment is resuspended in the remaining fluid and transferred to a fresh culture tube with medium and rice starch (and antibiotic if appropriate). After incubation for a further 48 hours the culture is reexamined as above. If no amebae are seen the subculture method is identical except that the size of the inoculum will be gradually reduced as the numbers of amebae increase.
Initially, it is usually helpful to centrifuge the cultures (275xg, 3 minutes) and split the pellet among the recipient tubes. This can be done by chilling the culture tube, inverting several times to detach adherent amebae, and transferring the liquid phase to an empty culture tube before pelleting (cultures in LE medium can be pelleted in situ, but in our experience the agar slant of Robinson's medium is not as conducive to this approach). As growth improves, pelleting is no longer necessary as measured inocula (< 2 ml) can be transferred to the fresh medium.
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