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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