Balamuthia mandrillaris Visvesvara et al. (ATCC® 50209)

Strain Designations: CDC:V039  /  Depositor: GS Visvesvara  /  Biosafety Level: 2

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Strain Designations CDC:V039
Biosafety Level 2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Isolation
brain tissue from 3 year, 10 month old female mandrill, Papio sphinx, that died of amebic meningoencephalitis, San Diego Zoo, 1986
Product Format frozen
Type Strain yes
Comments
growth in axenic media
The amoebae can be maintained on a number of different cell lines
agent of amebic meningoencephalitis
experimental infection in mice
Medium ATCC® Medium 1156: RP medium
Growth Conditions
Temperature: 35.0°C
Protocol: ATCCNO: 50209 SPEC: Upon arrival place frozen ampule directly into a 35C water bath, transfer thawed contents to 10 ml of fresh medium in a T-25 tissue culture flask containing a monolayer of African green monkey kidney cells (ATCC CRL-1586). The amoebae will completely destroy the monolayer and then encyst. Vigorously agitate the encysted culture and aseptically transfer 0.1 ml to a fresh monolayer of ATCC CRL-1586.
Subcultivation
Protocol: ATCCNO: 50209 SPEC: Upon arrival place frozen ampule directly into a 35C water bath, transfer thawed contents to 10 ml of fresh medium in a T-25 tissue culture flask containing a monolayer of African green monkey kidney cells (ATCC CRL-1586). The amoebae will completely destroy the monolayer and then encyst. Vigorously agitate the encysted culture and aseptically transfer 0.1 ml to a fresh monolayer of ATCC CRL-1586.
Cryopreservation

1.   To harvest the Balamuthia culture, detach cysts by scraping the inside bottom surface of the flask with a sterile cell scraper.

2.   Transfer the cyst suspension to 15 ml plastic centrifuge tubes. Centrifuge at approximately 800 x g for 5 min.

3.   Remove all but 0.5 ml of the supernatant from each tube, resuspend the cyst pellets, and pool them to a single tube.

4.   Adjust the concentration of cysts to 2.0 - 4.0 x 105 cysts/ml with fresh medium or PBS.

      NOTE: If the concentration of cysts is too low, centrifuge at 800 x g for 5 min and resuspend in the volume of fresh medium or PBS required to yield the desired concentration.

5.   Prepare a cryoprotective solution containing 15% (v/v) DMSO and 6% (v/v) HIFBS in fresh medium or PBS.

6.   Mix the cyst preparation and cryoprotective solution in equal portions. The final concentration will be 1.0 - 2.0 x 105 cysts/ml, 7.5% DMSO, and 3% HIFBS. The time from the mixing of the cyst preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.

7.   Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

8.   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. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)

9.   Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.

10.          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.

11.          Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of cells (ATCC CRL-1586 or CCL-81) and 10 ml ATCC 30-2003 with 3% (v/v) HIFBS.

12.          Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.

13.          Incubate in a 35°-37°C CO2 incubator with the cap screwed on tightly.

Name of Depositor GS Visvesvara
Year of Origin 1986
References

Visvesvara GS, et al. Balamuthia mandrillaris, n. g., n. sp., agent of amebic meningoencephalitis in humans and other animals. J. Eukaryot. Microbiol. 40: 504-514, 1993. PubMed: 8330028

Visvesvara GS, et al. Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals. J. Clin. Microbiol. 28: 2750-2756, 1990. PubMed: 2280005

Schuster FL, Visvesvara GS. Axenic growth and drug sensitivity studies of Balamuthia mandrillaris, an agent of amebic meningoencephalitis in humans and other animals. J. Clin. Microbiol. 34: 385-388, 1996. PubMed: 8789020

Rideout BA, et al. Fatal infections with Balamuthia mandrillaris (a free-living amoeba) in gorillas and other Old World primates. Vet. Pathol. 34: 15-22, 1997. PubMed: 9150541

Janitschke K, et al. Animal model Balamuthia mandrillaris CNS infection: contrast and comparison in immunodeficient and immunocompetent mice: a murine model of "granulomatous" amebic encephalitis. J. Neuropathol. Exp. Neurol. 55: 815-821, 1996. PubMed: 8965096

type strain

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation