Dissolve in this order in 600 ml deionized or glass distilled water:
Bring final volume to 880 ml and pH to 6.8 using 1N Sodium Hydroxide solution (ca. 7.5 ml). Dispense in 88 ml amounts into 125 ml glass bottles and autoclave for 15 minutes at 121° C with 15 lbs. pressure. The sterile TYI base can be stored frozen at -20° C for several months.
Vitamin mix #18 (11):
An earlier version of this vitamin mixture is also available commercially (Sigma Aldrich).
Combine solutions 1 and 2, bring the final volume to 200 ml with distilled water and sterilize through a 0.22 µm filter. Store in 100 ml amounts at 4°C for up to 6 months. The solution is light sensitive. The above recipe provides enough vitamin mix #18 to make ten liters of complete TYI-S-33.
To complete TYI-S-33 medium for use in axenic cultivation add 2.0 ml of vitamin mix #18 and 10-15 ml of heat inactivated adult bovine serum to each 88 ml of TYI broth. Use complete medium within 7 days. Dispense into 16x125 mm borosilicate glass disposable culture tubes. The tubes should be filled to ca 80% capacity (including inoculum). In most cases 13 ml of TYI-S-33 per tube is the correct amount as inocula for axenic E. histolytica are generally small in established cultures. The % serum used varies among isolates. Fetal bovine serum is not acceptable as fetuin is toxic to the parasite. Sources of adult bovine serum can be found here.
This medium was developed as an alternative to TYI-S-33 due to difficulties in obtaining lots of casein digest peptone that would support adequate growth of E. histolytica. The recipe for YI-S is identical to that of TYI-S-33 except that casein digest peptone is replaced weight for weight by additional yeast extract, making the final concentration of yeast extract 3%. YI-S is not without its own problems as the lot of yeast extract used is crucial to successful cultivation using this medium.
The development of YI-S had one unexpected benefit. One characteristic of what were once known as 'non-pathogenic E. histolytica' isolates and are now known as E. dispar is that they are refractory to axenization by the classic technique for obtaining axenic E. histolytica cultures. One isolate of E. dispar has now been axenized by a different method, but it will only grow in YI-S, not in TYI-S-33 (3). Whether this is a general characteristic of this species, which is of significant diagnostic importance, remains to be seen.
In the course of developing YI-S, several combinations of Oxoid neutralised liver digest and yeast extract were studied. One of these, designated LYI-S-2 (liver digest, yeast extract, iron, serum), was found to result in growth equal to that in TYI-S-33. Intent on producing a medium with as few biological ingredients as possible, the medium containing only yeast extract, YI-S, was extensively tested and published. No difference in the ability of YI-S and LYI-S-2 to support growth of E. histolytica was observed (unpublished results). After publication of YI-S further testing within our laboratory and by others disclosed the fact that some lots of yeast extract would not support any growth of the ameba while with others growth was very poor. In the case of the latter it was found that substitution of a small amount of liver digest for an equal amount of yeast extract enhanced growth considerably. LYI-S-2 is recommended when a given lot of yeast extract will support some growth, though poorly, of E. histolytica. LYI-S-2 is identical to YI-S except that weight for weight it contains 0.5% neutralized liver digest and only 2.5% yeast extract. It has been tested with yeast extracts from a number of commercial sources. It has been used in the long term cultivation of E. histolytica HM-1:IMSS (over 2 years), several other isolates of E. histolytica and a number of other Entamoeba species (monoxenic and axenic E. dispar SAW760, axenic E. invadens IP-1, E. moshkovskii Laredo and FIC, and E. terrapinae M) with similar yields to the more widely used TYI-S-33 and YI-S.
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