Acanthamoeba pustulosa Pussard and Pons (ATCC® 50252)

Organism: Acanthamoeba pustulosa Pussard and Pons  /  Depositor: GS Visvesvara

Permits and Restrictions

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Strain Designations clone GE 3a
Biosafety Level 1
Isolation
derived from existing strain
Product Format freeze-dried
Type Strain no
Comments
The species may be a junior synonym of Acanthamoeba palestinensis
Medium ATCC® Medium 712: PYG w/ Additives
Growth Conditions
Temperature: 25.0°C
Duration: axenic
Protocol: ATCCNO: 30010 SPEC: This strain is distributed as a freeze-dried preparation. See the general procedures for opening a freeze-dried vial. Aseptically add 0.5 ml of ice cold medium containing 12% (w/v) sucrose to the freeze-dried inner shell vial. Once the culture is completely rehydrated, aseptically add 1 ml of ATCC medium 712 and distribute to a 16 X 125 mm plastic screw-capped test tube or a T-25 tissue culture flask containing 5.0 ml of the same medium. Incubate the test tube culture horizontally with the cap on tight. Trophozoites should be evident in 1-5 days.
Subcultivation
Protocol: ATCCNO: 30010 SPEC: This strain is distributed as a freeze-dried preparation. See the general procedures for opening a freeze-dried vial. Aseptically add 0.5 ml of ice cold medium containing 12% (w/v) sucrose to the freeze-dried inner shell vial. Once the culture is completely rehydrated, aseptically add 1 ml of ATCC medium 712 and distribute to a 16 X 125 mm plastic screw-capped test tube or a T-25 tissue culture flask containing 5.0 ml of the same medium. Incubate the test tube culture horizontally with the cap on tight. Trophozoites should be evident in 1-5 days.
Cryopreservation

1.   To achieve the best results set up cultures with several different inocula (e.g. 0.25 ml, 0.5 ml, 1.0 ml).  Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density.

2.  If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107cysts/ml with fresh medium.  If the concentration is too low, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.

3.  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.

4.  Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 106 and 107 cells/ml and 7.5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.

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

6.   Place the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at        -1°C/min through the heat of fusion.  At -40°C plunge 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.)  

7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.

8.   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 just sufficient to cover the frozen material. Do not agitate the vial.

9.   Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5 ml of fresh ATCC medium 712 in a T-25 tissue culture flask or plastic 16 x 125 mm screw-capped test tube.  Incubate at 25°C.

Name of Depositor GS Visvesvara
Chain of Custody
ATCC <<--GS Visvesvara<<--M. Pussard
References

. . J. Protozool. 30: 701-706, 1983.

Stothard DR, et al. The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types. J. Eukaryot. Microbiol. 45: 45-54, 1998. PubMed: 9495032

Stothard DR, et al. Fluorescent oligonucleotide probes for clinical and environmental detection of Acanthamoeba and the T4 18S rRNA gene sequence type. J. Clin. Microbiol. 37: 2687-2693, 1999. PubMed: 10405422

Kong HH, et al. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea. J. Clin. Microbiol. 40: 1199-1206, 2002. PubMed: 11923331

Gast RJ. Development of an Acanthamoeba-specific reverse dot-blot and the discovery of a new ribotype. J. Eukaryot. Microbiol. 48: 609-615, 2001. PubMed: 11831768

Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 16: 273-307, 2003. PubMed: 12692099

derived from strain GE3a

Cross References

Nucleotide (GenBank) : AF019050 18S ribosomal RNA gene, partial sequence.

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