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Reagents
CPMB-5 Cryoprotective Solution
DMSO, 1.0 mL
2.5 M Sucrose, 0.8 mL
L-Cysteine/Ascorbic Acid Solution, 0.2 mL
CPMB-2 Basal Solution, 6.0 mL
HIBS, 2.0 mL

CPMB-2 Basal Solution
Yeast Extract, 60.0 g
K₂HPO₄, 1.0 g
KH₂PO₄, 0.6 g
NaCl, 2.0 g
Distilled water, 1.0 L
Autoclave for 15 minutes.

L-Cysteine/Ascorbic Acid Solution
L-Cysteine-HCL, 1.0 g
Acorbic Acid, 0.1 g
Distilled water, 10.0 mL
Add 9.0 mL of distilled water to a 20 mL beaker and dissolve the first two components. While stirring, adjust the pH to 7.2 with 10N NaOH (approximately 0.7 mL). Adjust final volume to 10 mL with distilled water and filter sterilize. Solution should be used soon after preparation. Discard any unused solution.

Harvest and Preservation

1. Harvest cells from several cultures that 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 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 test 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 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 the vials in a controlled rate freezing unit.  Use the following cooling cycle: From room temperature cool at -10°C/min to the heat of fusion; from the heat of fusion to -40°C, cool at -1°C/min.   At -40°C plunge into liquid nitrogen.  The cooling cycle should be initiated no less than 15 and no more than 30 minutes after the addition of DMSO to the cell preparation.
10. Store ampules in a liquid nitrogen refrigerator until needed.
11. To establish a culture from the frozen state, place an ampule in a 25°C water bath, until thawed (2-3 min).  Immerse the vial just sufficiently to cover the frozen material.  Do not agitate the ampule.
12. Transfer contents of thawed ampule to a 16 x 125 mm screw-capped borosilicate glass test tube containing 13 mL of ATCC medium 2154.
13. Screw cap on tightly and incubate at a 15° horizontal slant at 35°C.  Observe the culture daily and transfer when many trophozoites are observed.

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

Gillin FD, Diamond LS. Clonal growth of Entamoeba histolytica and other species of Entamoeba in agar. J. Protozool. 25: 539-543, 1978. PubMed: 216801

Can. J. Zool. 36: 513-523, 1958.

Katiyar SK, Edlind TD. Entamoeba histolytica encodes a highly divergent beta-tubulin. J. Eukaryot. Microbiol. 43: 31-34, 1996. PubMed: 8563707

Manning-Cela R, et al. Actin mRNA levels and actin synthesis during the encystation of Entamobea invadens. J. Eukaryot. Microbiol. 41: 360-365, 1994. PubMed: 8087106

Diamond LS, Cunnick CC. A serum-free, partly defined medium, PDM-805, for axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and other Entamoeba. J. Protozool. 38: 211-216, 1991. PubMed: 1880759

Cho J, Eichinger D. Crithidia fasciculata induces encystation of Entamoeba invadens in a galactose-dependent manner. J. Parasitol. 84: 705-710, 1998. PubMed: 9714198

Ehrenkaufer GM, et al. The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation. Genome Biol. 14(7) :R77, 2013. PubMed: 23889909

Nucleotide (GenBank) : L29559 Entamoeba invadens (clone EIRD1234) small subunit ribosomal RNA gene, 3' end; 5.8S ribosomal RNA gene, complete sequence; large subunit ribosomal RNA gene, 5'end.

Nucleotide (GenBank) : AANW02000000 Entamoeba invadens IP1, whole genome shotgun sequencing project.