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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.
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Invert tubes 20 times and centrifuge at 200-300 x g for 5 min.
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While cells are centrifuging, prepare the cryoprotective solution.
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Place 1.0 mL of DMSO in a 16 x 125 mm screw-capped tube and ice until solidified.
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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.
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Add 0.2 mL of the L-Cysteine/Ascorbic Acid solution to the DMSO solution and mix.
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Add 6.0 mL of the CPMB-2 Basal Solution and mix.
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Add 2.0 mL HIBS (heat-inactivated bovine serum) and mix.
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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.
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After the cell concentration is adjusted, centrifuge as in step 2.
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Remove as much supernatant as possible and determine the volume removed.
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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.
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Dispense 0.5 mL aliquots into 1.0 - 2.0 mL plastic sterile cryules (special plastic vials for cryopreservation).
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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.
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Store ampules in a liquid nitrogen refrigerator until needed.
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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.
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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.
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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.
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Transfer contents of the thawed ampule to a one-dram screw-capped vial (vial holds approximately 4.0 mL).
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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.
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Ice the vial for 10 minutes, then invert gently 10 times. Centrifuge the vial at 100-200 x g for 5 min.
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Aspirate the supernatant leaving approximately 0.5 mL. Note: Do not aspirate the pelleted material.
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Replace the supernatant with 3.0 mL of the bacterized medium 1171 prepared in step 11.
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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).
