ch, 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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