Acanthamoeba polyphaga (Puschkarew) Page (ATCC® 50998)

Organism: Acanthamoeba polyphaga (Puschkarew) Page  / 

Permits and Restrictions

View Permits

Strain Designations LNKY-1
Biosafety Level 2
Isolation
United Kingdom
Product Format frozen
Type Strain no
Medium ATCC® Medium 712: PYG w/ Additives
Growth Conditions
Temperature: 25.0°C
Duration: Axenic
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.

Mycoplasma No
Special Collection NCRR Contract
References

Gao LY, et al. Identification of macrophage-specific infectivity loci (mil) of Legionella pneumophila that are not required for infectivity of protozoa. Infect. Immun. 66: 883-892, 1998. PubMed: 9488371

Harb OS, et al. Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts. Appl. Environ. Microbiol. 64: 126-132, 1998. PubMed: 9435069

Harb OS, Abu Kwaik Y. Identification of the aspartate-beta-semialdehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant. Infect. Immun. 66: 1898-1903, 1998. PubMed: 9573067

Stone BJ, Abu Kwaik Y. Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect. Immun. 66: 1768-1775, 1998. PubMed: 9529112

Pedersen LL, et al. HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells. Infect. Immun. 69: 2569-2579, 2001. PubMed: 11254621

Viswanathan VK, et al. The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element. Infect. Immun. 70: 1842-1852, 2002. PubMed: 11895946

Gao L-Y, et al. Heterogeneity in intracellular replication and cytopathogenicity of Legionella pneumophila and Legionella micdadei in mammalian and protozoan cells. Microb. Pathog. 27: 273-287, 1999. PubMed: 10545255

Gao LY, et al. Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant host cells, mammalian macrophages and protozoa. Infect. Immun. 65: 4738-4746, 1997. PubMed: 9353059

Molmeret M, et al. The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga. Mol. Microbiol. 43: 1139-1150, 2002. PubMed: 11918802

Molmeret M, et al. icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infect. Immun. 70: 69-78, 2002. PubMed: 11748165

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