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Entamoeba dispar Brumpt

PRA-368

Product category
Protists
Classification
Amoebozoa, Entamoebida,
Strain designation
NIH:0581:1
Type strain
No
Product format
Frozen
Storage conditions
-80°C or colder for 1 week, vapor phase of liquid nitrogen for long-term storage
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Documentation

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

Handling information

Medium
Instruction for complete medium
Grown with mixed bacteria. Quality controlled lots of this medium are commercially available from ATCC as cat. no. PRA-1171™.
Temperature
35°C
Atmosphere
Microaerophilic
Culture system
Xenic
Handling procedure
Storage and Culture Initiation
Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen.  If liquid nitrogen storage facilities are not available, frozen ampules may be stored at or below -70°C for approximately one week.  Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C).  Storage of frozen material at this temperature will result in the death of the culture.
  1. One day before thawing a frozen ampule inoculate two tubes of ATCC medium 1171 with the bacterial flora only.  Incubate the tubes on a 15° horizontal slant at 35°C.  If the specific bacterial flora associated with this culture are not available, skip this step and proceed to step 2.
  2. On the following day combine 4.1 mL ATCC medium 1171 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.  Note:  If bacterized medium 1171 was prepared in step 1, use the bacterized medium in place of fresh medium for faster recovery/growth.
  3. 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 2.  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.
  4. Transfer contents of the thawed ampule to a one-dram screw-capped vial (vial holds approximately 4.0 mL).
  5. Add 2.5 mL of serum-enriched medium prepared in step 2 to the vial in dropwise fashion.  Tighten the cap and incubate on a 15° horizontal slant at 35°C for 2-3 hours.
  6. Ice the vial for 10 minutes, then invert gently 10 times. Centrifuge the vial at 100-200 x g for 5 min.
  7. Aspirate the supernatant leaving approximately 0.5 mL.  Note:  Do not aspirate the pelleted material. 
  8. Replace the supernatant with 3.0 mL of the bacterized medium 1171 prepared in step 1 if available; otherwise use fresh medium 1171 (i.e., not serum-enriched).
  9. Incubate the vial on a 15° horizontal slant at 35°C with the cap screwed on tightly.  Observe the culture daily and transfer when many trophozoites are observed (i.e., early stationary phase).
Handling notes
This xenic culture contains the original bacterial flora present when the amoeba was first isolated.
Culture maintenance
  1. Ice a test tube culture at or near peak density for 10 minutes, invert 20 times and aseptically transfer a 0.1 and 0.3 mL aliquot to a fresh tube of ATCC medium 1171.
  2. Screw cap on tightly and incubate on a 15° horizontal slant at 35°C.  Transfer when many trophozoites are observed (i.e., early stationary phase).

Notes:  In general, better growth can often be achieved by using media pre-inoculated with bacteria.  Addition of penicillin G at 75 U/mL and streptomycin at 75 µg/mL to ATCC Medium 1171 may be necessary if the bacterial density in the culture is too high.  Inoculate fresh tubes of media with bacteria at least one day prior to subcultivation and prior to addition of antibiotics, if used.  Also, nutrients in ATCC medium 1171 may be diluted through use of an osmotically-balanced saline buffer such as Dulbecco's PBS (ATCC 30-2200) with the medium.  Mixtures in the range of 1:1 to 3:1 (medium to buffer) usually work well to control or limit bacterial growth in cultures where other methods are not sufficient.

Reagents for cryopreservation
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.
Cryopreservation
  1. 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.
  2. Invert tubes 20 times and centrifuge at 200-300 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 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 (heat-inactivated bovine serum) 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 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.
  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.  Incubate the tube on a 15° horizontal slant at 35°C.  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.  Invert gently several times to mix.
  13. 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.
  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.  Tighten the cap and incubate on a 15° horizontal slant at 35°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.  Note:  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 35°C with the cap screwed on tightly.  Observe the culture daily and transfer when many trophozoites are observed (i.e., early stationary phase).

History

Deposited as
Entamoeba histolytica Schaudinn
Depositors
LS Diamond
Type of isolate
Human

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Clark CG, Diamond LS. Ribosomal RNA genes of 'pathogenic' and 'nonpathogenic' Entamoeba histolytica are distinct. Mol. Biochem. Parasitol. 49: 297-302, 1991. PubMed: 1685555

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

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