Entamoeba moshkovskii Tshalaia

1. Remove 5.5 mL of fluid and centrifuge at 500 x g for 5 minutes.
2. Inoculate two fresh tubes of ATCC medium 1171 with 0.25 mL of supernatant each from the fluid centrifuged in step 1 (preinoculated bacterized culture tubes may allow for better growth).
3. Divide the remainder of the supernatant in two equal aliquots in 16 x 125 mm screw-capped tubes and bring the volumes up to 8 mL with fresh ATCC Medium 1171.
4. Ice the parent culture 5 minutes, invert 20 times and transfer 0.5 and 1.0 mL aliquots to the test tubes containing the equal volumes of supernatant.
5. Re-feed the parent culture by centrifuging it at 200 x g for 5 min, aspirate most of the supernatant (leaving approximately 1.0-1.5 mL), and resuspend the pellet with fresh growth medium up to 8 mL.
6. Incubate all cultures (including the tubes of bacterized ATCC medium 1171) at a 15° horizontal slant at 25°C.
7. Observe the culture daily and transfer when many trophozoites are observed (i.e., early stationary phase).

1. Ice a test tube culture at or near peak density for 10 minutes, invert 20 times and aseptically transfer a 0.1 and 0.3 mL aliquot to a fresh tube of ATCC medium 1171.
2. Screw cap on tightly and incubate on a 15° horizontal slant at 25°C.  Transfer when many trophozoites are observed (i.e., early stationary phase).

Notes:  In general, better growth can often be achieved by using media pre-inoculated with bacteria.  Addition of penicillin G at 75 U/mL and streptomycin at 75 µg/mL to ATCC Medium 1171 may be necessary if the bacterial density in the culture is too high.  Inoculate fresh tubes of media with bacteria at least one day prior to subcultivation and prior to addition of antibiotics, if used.  Also, nutrients in ATCC medium 1171 may be diluted through use of an osmotically-balanced saline buffer such as Dulbecco's PBS (ATCC 30-2200) with the medium.  Mixtures in the range of 1:1 to 3:1 (medium to buffer) usually work well to control or limit bacterial growth in cultures where other methods are not sufficient.

1. Harvest cells from several cultures which are in the late logarithmic to early stationary phase of growth.  Place culture vessels on ice for 10 min.
2. Invert tubes 20 times and centrifuge at 200-300 x g for 5 min.        
3. While cells are centrifuging, prepare the cryoprotective solution. 
   1. Place 1.0 mL of DMSO in a 16 x 125 mm screw-capped tube and ice until solidified.
   2. Add 0.8 mL of the 2.5 M Sucrose solution, remove from ice and invert until the DMSO is liquefied.  Return to ice bath.
   3. Add 0.2 mL of the L-Cysteine/Ascorbic Acid solution to the DMSO solution and mix.
   4. Add 6.0 mL of the CPMB-2 Basal Solution and mix.
   5. Add 2.0 mL HIBS (heat-inactivated bovine serum) and mix.
4. Resuspend the cell pellets and pool to a final volume of approximately 10 mL with the supernatant.  Make a determination of the cell density and adjust the concentration of the cells between 5 x 10⁵/mL - 1 x 10⁶/mL using fresh medium.  If the cell concentration is below 5 x 10⁵/mL, centrifuge the cell suspension and resuspend the pellet in a volume that will yield the desired concentration.
5. After the cell concentration is adjusted, centrifuge as in step 2.
6. Remove as much supernatant as possible and determine the volume removed.
7. Resuspend the cell pellet with a volume of the cryoprotective solution equal to the volume of the supernatant removed.  Invert the tube several times to obtain a uniform cell density.
8. Dispense 0.5 mL aliquots into 1.0 - 2.0 mL plastic sterile cryules (special plastic vials for cryopreservation).
9. Place vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion.  At -40°C plunge ampules into liquid nitrogen.
10. Store ampules in a liquid nitrogen refrigerator until needed.
11. One day before thawing a frozen ampule inoculate two tubes of ATCC medium 1171 with the bacterial flora only.  Incubate the tube on a 15° horizontal slant at 25°C.  If the specific bacterial flora associated with this culture are not available, skip this step and proceed to step 12.
12. On the following day combine 4.1 mL of the bacterized medium 1171 prepared in step 11 with 0.9 mL of HIBS (heat-inactivated bovine serum) to produce 5 mL of medium enriched with 20% serum.  Invert gently several times to mix.
13. Remove the frozen ampule from liquid nitrogen and flame gently at the base of the cap.  Remove the cap and aseptically add 0.5 mL of the serum-enriched medium prepared in step 12.  Place in a 35°C water bath until thawed (2-3 min).  Note:  Manipulations of the ampule before placing in the water bath should be done as quickly as possible to avoid warming of the contents at a suboptimal rate.
14. Transfer contents of the thawed ampule to a one-dram screw-capped vial (vial holds approximately 4.0 mL).
15. Add 2.5 mL of serum-enriched medium prepared in step 12 to the vial in dropwise fashion.  Tighten the cap and incubate on a 15° horizontal slant at 25°C for 2-3 hours.
16. Ice the vial for 10 minutes, then invert gently 10 times. Centrifuge the vial at 100-200 x g for 5 min.
17. Aspirate the supernatant leaving approximately 0.5 mL.  Note:  Do not aspirate the pelleted material.
18. Replace the supernatant with 3.0 mL of the bacterized medium 1171 prepared in step 11.
19. Incubate the vial on a 15° horizontal slant at 25°C with the cap screwed on tightly.  Observe the culture daily and transfer when many trophozoites are observed (i.e., early stationary phase).

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