Entamoeba histolytica Schaudinn (ATCC® 30015)

Strain Designations: HK-9  /  Depositor: LS Diamond  /  Biosafety Level: 2

Strain Designations HK-9
Application
Enteric Research
Food and waterborne pathogen research
Biosafety Level 2
Isolation Colonic biopsy from adult human male with amebic dysentery, Korea, (?)
Product Format frozen
Storage Conditions Frozen Cultures:
-70°C for 1 week; liquid N2 vapor for long term storage

Freeze-dried Cultures:
2-8°C

Live Cultures:
See Protocols section for handling information
Type Strain no
Comments Entamoeba phylogeny
Zymodeme II
Not detected by Trichomonas vaginalis-specific PCR primers
Axenic cultivation
Use of plastic ampoules for freeze preservation
Growth responses to reducing agents
Effect of cysteine and oxygen tension on survival
Requirement for cysteine and ascorbic acid for attachment
Medium ATCC® Medium 2154: LYI Entamoeba medium
Growth Conditions Temperature: 35°C
Atmosphere: Anaerobic
Culture System: Axenic
Cryopreservation 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 10 M 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 to 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 mL to 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:
    1. From room temperature cool at -10°C/min to the heat of fusion
    2. From the heat of fusion to -40°C, cool at -1°C/min
    3. 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 to 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.
Mycoplasma No
Name of Depositor LS Diamond
Special Collection NCRR Contract
References

Charoenlarp P, et al. Carbohydrate utilization by Entamoeba histolytica. Exp. Parasitol. 23: 205-211, 1968. PubMed: 4302387

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

Diamond LS. Axenic cultivation of Entamoeba histolytica. Science 134: 336-337, 1961. PubMed: 13722605

Diamond LS. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. J. Parasitol. 54: 1047-1056, 1968. PubMed: 4319346

Garcia LS, et al. Comparison of indirect fluorescent-antibody amoebic serology with counterimmunoelectrophoresis and indirect hemagglutination amoebic serologies. J. Clin. Microbiol. 15: 603-605, 1982. PubMed: 6279694

Gelderman AH, et al. Characterization of the deoxyribonucleic acid of representative strains of Entamoeba histolytica, E. histoytica-like amebae, and E. moshkovskii. J. Parasitol. 57: 906-911, 1971. PubMed: 4327966

Gelderman AH, et al. A comparison of genome sizes and thermal-denaturation-derived base composition of DNAs from several members of Entamoeba (histolytica group). J. Parasitol. 57: 912-916, 1971. PubMed: 4327967

Gillin FD, Diamond LS. Attachment of Entamoeba histolytica to glass in a defined maintenance medium: Specific requirement for cysteine and ascorbic acid. J. Protozool. 27: 474-478, 1980. PubMed: 6260930

Gillin FD, Diamond LS. Entamoeba histolytica and Giardia lamblia: growth responses to reducing agents. Exp. Parasitol. 51: 382-391, 1981. PubMed: 6262103

Gillin FD, Diamond LS. Entamoeba histolytica and Giardia lamblia: effects of cysteine and oxygen tension on trophozoite attachment to glass and survival in culture media. Exp. Parasitol. 52: 9-17, 1981. PubMed: 6263667

Gonzalez-Ruiz A, et al. Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay. J. Clin. Microbiol. 32: 964-970, 1994. PubMed: 8027351

Madico G, et al. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples. J. Clin. Microbiol. 36: 3205-3210, 1998. PubMed: 9774566

Phillips BP, et al. Results of intracecal inoculation of germfree and conventional guinea pigs and germfree rats with axenically cultivated Entamoeba histolytica. J. Protozool. 19: 498-499, 1972. PubMed: 4342299

Procedures for the Recovery and Identification of Parasites from the Intestinal Tract: Approved Guideline - 2nd Edition. Wayne, PA. Clinical and Laboratory Standards Institute; CLSI M28-A2.

Simione FP Jr., et al. The use of plastic ampoules for freeze preservation of microorganisms. Cryobiology 14: 500-502, 1977. PubMed: 891238

Takeuchi T, et al. Entamoeba histolytica: localization and characterization of phosphorylase and particulate glycogen. Exp. Parasitol. 43: 107-114, 1977. PubMed: 196880

Takeuchi T, et al. Pyruvate oxidase (CoA acetylating) in Entamoeba histolytica. Biochem. Biophys. Res. Commun. 65: 591-596, 1975. PubMed: 167776