Acanthamoeba sp. (ATCC® PRA-228)

Organism: Acanthamoeba sp.  /  Depositor: S Schmitz-Esser

Permits and Restrictions

View Permits

Strain Designations 5a2
Biosafety Level 2
Isolation
Lake sediment, Lake Neusiedl, Burgenland, Austria
Product Format frozen
Type Strain no
Comments
Genome sequence of the symbiont Candidatus Amoebophilus asiaticus
Medium ATCC® Medium 663: PYG medium
Growth Conditions
Temperature: 20.0°C
Growth condition: axenic
Protocol: This strain is shipped frozen. Thaw the frozen material in a 35C water bath and transfer the contents into a T25 flask containing 10ml of growth medium. Monitor for up to 3-4 weeks until the culture has reached peak density. To subculture, shake flask and transfer one tenth of the culture into a new flask with fresh medium.
Cryopreservation
Storage temperature: liquid nitrogen vapor phase

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 30°C.

Cryoprotective solution: 7.5% DMSO in growth medium mixed with Dryl's solution (ATCC medium 5080) at a 1:1 ratio.
Name of Depositor S Schmitz-Esser
References

Schmitz-Esser S, et al. Diversity of bacterial endosymbionts of environmental acanthamoeba isolates. Appl. Environ. Microbiol. 74: 5822-5831, 2008. PubMed: 18641160

Schmitz-Esser S, et al. The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" reveals common mechanisms for host cell interaction among amoeba-associated bacteria. J. Bacteriol. 192: 1045-1057, 2010. PubMed: 20023027

Cross References

Nucleotide (GenBank) : CP001102 Candidatus Amoebophilus asiaticus 5a2, complete genome

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