Established cultures of all three parasites are essentially handled in the same way.
Xenic cultures of E. histolytica are routinely passaged at 48-72 hour intervals; usually a Monday/ Wednesday/ Friday schedule is convenient. The inoculum size for the longer incubation period should be smaller than that for shorter incubations. However, variation among isolates and flora means that no generalities can be made regarding the size of inocula or the amount of rice and antibiotics to be added to the medium for optimal growth. It is very much a case of trial and error combined with experience in evaluating growth of cultures that leads to successful establishment of E. histolytica in xenic culture. It is recommended that xenic cultures be passaged using two or more inoculum sizes to ensure a successful subculture. A significant threshold effect can sometimes be encountered, where an certain inoculum size gives rise to a healthy culture but an inoculum smaller by as little as 50 µl may result in no amebal growth.
Axenic cultures of E. histolytica are passaged at 72 and 96 hour intervals, with a Monday/ Friday schedule being convenient. Again the inocula for the longer incubation period should be smaller than for the shorter, and again trial and error is needed to find out exactly what those are, due to significant variation among isolates. While counted inocula are desirable, established cultures become predictable and measured volume inocula can be used.
Visual inspection of every culture before subculture is recommended since what appears to be a heavy culture may in fact contain many lysed cells, indicating that the previous inoculum used was too large. An increased inoculum volume may be warranted for the following subculture to compensate for the dead amebae. Likewise, parallel duplicate cultures are recommended in case of inadvertent contamination or tube breakage. The unused culture can be kept at 33° C as a 'backup' in case of problems.
The method for subculturing all types of cultures is essentially the same. Cultures are chilled in an ice-water bath for 5 minutes (both xenic and axenic E. histolytica) to release trophozoites attached to the glass culture tube. Chilled tubes are inverted several times to disperse the cells and a measured inoculum is passed aseptically to a culture tube containing 13 ml of fresh medium. The tubes are capped tightly and incubated at 35.5° C, either vertically (xenic cultures) or at 5° to the horizontal (established axenic cultures).