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Echinamoeba exundans Page

50171

Product category
Protists
Classification
Amoebozoa, Tubulinea, Incertae sedis Tubulinea
Strain designation
SH274
Type strain
No
Isolation source
Hot water tank
Geographical isolation
United States; California
Product format
Frozen
Storage conditions
-80°C or colder for 1 week, vapor phase of liquid nitrogen for long-term storage
Mission Collection Item
This is a Mission Collection Item

Documentation

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Biosafety Icon BSL 1

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

Characteristics

Comments
Intracellular multiplication of Legionella pneumophila

Handling information

Medium
Instruction for complete medium
ATCC Medium 711; grown with mixed bacteria
Temperature
25°C
Culture system
Xenic
Incubation
Grown with mixed bacteria
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 ampoules 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. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.  Do not leave ampule in water bath after thawed.
  2. Immediately after thawing, aseptically transfer contents to the ATCC medium 711 plate.
  3. Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.  Trophozoites should be seen within 2-3 d.
Culture maintenance
  1. Streak an ATCC medium 711 plate with Enterobacter aerogenes (ATCC® 13048™) and incubate at 35°C overnight.
  2. Remove an agar block (~5 mm2 ), with trophozoites or cysts, from the edge of an agar plate culture and invert the block at the edge of the freshly bacterized plate.
  3. Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.
  4. Repeat steps 1-3 at 10-14 d intervals.

  5. Note: a monoxenic amoeba culture can be established in this manner using any suitable bacterial food source.
Cryopreservation
  1. Harvest cells from a culture which is at or near peak density by adding 5 mL fresh ATCC medium 1323 (Page's Balanced Salt Solution) and washing cells into suspension.  Rub the surface of the plate with a spread bar to detach adhering amoebae.
  2. Transfer the liquid medium to a sterile centrifuge tube.
  3. If the cell concentration does not exceed 2 x 106 cells/mL adjust the suspension to that concentration.  To adjust the concentration, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield 2 x 106.
  4. While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.  Allow the DMSO to solidify.  Add the required volume of refrigerated medium.  Dissolve the DMSO by inverting the tube several times.
    *NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
  5. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be at least 106 cells/mL and 7.5% (v/v) DMSO.  The equilibration time  (the time between addition of DMSO  and the start of  the cooling cycle) should be no less than 15 min and no longer than 60 min.
  6. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
  7. 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.
  8. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
  9. To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
  10. Immediately after thawing, aseptically remove the contents of the ampule and distribute to the center of a fresh plate of ATCC medium 711.  Distribute the material evenly over the plate using a spread bar.  Incubate at 25°C.

History

Deposited as
Echinamoeba exundans Page
Depositors
BS Fields
Year of origin
1987

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

Fields BS, et al. Intracellular multiplication of Legionella pneumophila in amoebae isolated from hospital hot water tanks. Curr. Microbiol. 18: 131-137, 1989.

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