1. Harvest cells from several cultures which are in the late logarithmic to early stationary phase of growth.xa0 Place culture vessels on ice for 10 min.
2. Invert tubes 20 times and centrifuge at 200-300 x g for 5 min.xa0xa0xa0xa0xa0xa0xa0xa0
3. While cells are centrifuging, prepare the cryoprotective solution.xa0
   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.xa0 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.xa0 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.xa0 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.xa0 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.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion.xa0 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.xa0 Incubate the tube on a 15° horizontal slant at 25°C.xa0 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.xa0 Invert gently several times to mix.
13. Remove the frozen ampule from liquid nitrogen and flame gently at the base of the cap.xa0 Remove the cap and aseptically add 0.5 mL of the serum-enriched medium prepared in step 12.xa0 Place in a 35°C water bath until thawed (2-3 min).xa0 Note:xa0 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.xa0 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.xa0 Note:xa0 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.xa0 Observe the culture daily and transfer when many trophozoites are observed (i.e., early stationary phase).

Clark CG, Diamond LS. Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting. J. Eukaryot. Microbiol. 44: 142-154, 1997. PubMed: 9109261