Acanthamoeba castellanii (Douglas) Page (ATCC® 50739)

Organism: Acanthamoeba castellanii (Douglas) Page  /  Depositor: R Michel

Permits and Restrictions

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Strain Designations C3
Application
Preferred host for the bacterial parasite Parachlamydia acanthamoebae ATCC VR-1476
Biosafety Level 2
Product Format frozen
Type Strain no
Comments
Preferred host for the bacterial parasite Parachlamydia acanthamoebae ATCC VR-1476
Medium Medium 1021: SCGYEM medium
Medium 712: PYG w/ Additives
Growth Conditions
Temperature: 20-30.0°C
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 10 ml of fresh ATCC medium 1021 in a T-25 tissue culture flask or 5 ml fresh ATCC medium 1021 in a 16 x 125 mm screw-capped plastic test tube.  Incubate at 20-25°C (incubate test tubes at a 15° horizontal slant).

Name of Depositor R Michel
Special Collection NCRR Contract
References

Amann R, et al. Obligate intracellular bacterial parasites of Acanthamoebae related to Chlamydia spp.. Appl. Environ. Microbiol. 63: 115-121, 1997. PubMed: 8979345

Michel R, et al. Legionella-like slender rods multiplying within a strain of Acanthamoeba sp. isolated from drinking water. Parasitol. Res. 84: 84-88, 1998. PubMed: 9491433

Marolda CL, et al. Intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae. Microbiology 145: 1509-1517, 1999. PubMed: 10439391

Birtles RJ, et al. 'Candidatus Odyssella thessalonicensis' gen. nov., sp. nov., an obligate intracellular parasite of Acanthamoeba species. Int. J. Syst. Evol. Microbiol. 50: 63-72, 2000. PubMed: 10826788

Michel R, et al. Enlarged Chlamydia-like organisms as spontaneous infection of Acanthamoeba castellanii. Parasitol. Res. 87: 248-251, 2001. PubMed: 11293575

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