Entamoeba histolytica Schaudinn (ATCC^® 30459^™)

Yes

 
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
Ascorbic 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 35°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 2463.
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.   

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Landa L, et al. Advances in methods of Entamoeba histolytica culture. Arch. Invest. Med. 1 suppl.: 9-14, 1970.

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

Bakker-Grunwald T, et al. Characterization of glycogen and amino acid pool of Entamoeba histolytica by C-NMR spectroscopy. J. Eukaryot. Microbiol. 42: 346-349, 1995. PubMed: 7620458

Que X, Reed SL. Nucleotide sequence of a small subunit ribosomal RNA (16S-like rRNA) gene from Entamoeba histolytica: differentiation of pathogenic from nonpathogenic isolates. Nucleic Acids Res. 19: 5438, 1991. PubMed: 1923831

Reed SL, et al. Thiol proteinase expression and pathogenicity of Entamoeba histolytica. J. Clin. Microbiol. 27: 2772-2777, 1989. PubMed: 2556432

Long-Krug SA, et al. Phospholipase A enzymes of Entamoeba histolytica: description and subcellular localization. J. Infect. Dis. 152: 536-541, 1985. PubMed: 2863316

Mirelman D, et al. Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence. Exp. Parasitol. 62: 142-148, 1986. PubMed: 2873049

Li E, et al. Interaction of laminin with Entamoeba histolytica cysteine proteinases and its effect on amebic pathogenesis. Infect. Immun. 63: 4150-4153, 1995. PubMed: 7558332

Ravdin JI, et al. Effect of antagonists of calcium and phospholipase A on the cytopathogenicity of Entamoeba histolytica. J. Infect. Dis. 152: 542-549, 1985. PubMed: 2863317

Li E, et al. Entamoeba histolytica interactions with polarized human intestinal Caco-2 epithelial cells. Infect. Immun. 62: 5112-5119, 1994. PubMed: 7927794

Torian BE, et al. The 96-kilodalton antigen as an integral membrane protein in pathogenic Entamoeba histolytica: potential differences in pathogenic and nonpathogenic isolates. Infect. Immun. 58: 753-760, 1990. PubMed: 2307518

Reed SL, et al. Molecular and cellular characterization of the 29-kilodalton peripheral membrane protein of Entamoeba histolytica: differentiation between pathogenic and nonpathogenic isolates. Infect. Immun. 60: 542-549, 1992. PubMed: 1730488

Mattern CF, et al. Entamoeba histolytica 'toxin' fetuin neutralizable and lectin-like. Am. J. Trop. Med. Hyg. 29: 26-30, 1980. PubMed: 6153255

Flores BM, et al. Surface localization, regulation, and biologic properties of the 96-kDa alcohol/aldehyde dehydrogenase (EhADH2) of pathogenic Entamoeba histolytica. J. Infect. Dis. 173: 226-231, 1996. PubMed: 8537663

Sanchez LB. Cloning and heterologous expression of Entamoeba histolytica adenylate kinase and uridylate/cytidylate kinase. Gene 209: 219-228, 1998. PubMed: 9524270

Pillai DR, et al. The cysteine-rich region of the Entamoeba histolytica adherence lectin (170-kilodalton subunit) is sufficient for high-affinity Gal/GalNAc-specific binding in vitro. Infect. Immun. 67: 3836-3841, 1999. PubMed: 10417146

del Aguila C, et al. Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis. J. Clin. Microbiol. 35: 1862-1866, 1997. PubMed: 9196210

Adagu IS, et al. In vitro activity of nitazoxanide and related compounds against isolates of Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. J. Antimicrob. Chemother. 49: 103-111, 2002. PubMed: 11751773

, with additional 5% HIBS added

Sanchez LB, et al. Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships. Gene 295: 51-59, 2002. PubMed: 12242011

Nucleotide (GenBank) : X98567 E. histolytica ubi1 gene.

Nucleotide (GenBank) : M77240 Entamoeba histolytica EHZc3 protein gene, complete cds.

Nucleotide (GenBank) : M80910 Entamoeba histolytica serine-rich protein (SREHP) gene, complete cds.

Nucleotide (GenBank) : U67157 Entamoeba histolytica 30 kDa type I collagen binding protein mRNA, partial cds.

Nucleotide (GenBank) : U67158 Entamoeba histolytica 30 kDa type I collagen binding protein mRNA, partial cds.

Nucleotide (GenBank) : AAFB02000000 Entamoeba histolytica HM-1:IMSS, whole genome shotgun sequencing project.