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
Hospital cooling tower, Lyon, France, 2007
Product Format
frozen
Storage Conditions
Frozen Cultures: -70°C for 1 week; liquid N2 vapor for long term storage
Freeze-dried Cultures: 2-8°C
Live Cultures: See Protocols section for handling information
Type Strain
yes
Comments
This axenic culture must be fed with heat-killed Escherichia coli bacteria (ATCC® 23740™ or similar).
Medium
ATCC® Medium 1034: Modified PYNFH medium (Available from ATCC as ATCC cat. no. 327-X)
Growth Conditions
Temperature: 20°C to 25°C Atmosphere: Aerobic Culture System: Axenic
Cryopreservation
Reagents Cryoprotective Solution
DMSO, 1.5 mL
Fresh complete growth medium, 8.5 mL
Harvest and Preservation
To achieve the best results, set up cultures with several different inocula (i.e., 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.
If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107cells/mL with fresh growthmedium. If the concentration is too low, centrifuge at 1200 to 1600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
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.
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 to the start of the freezing process should be no less than 15 min and no longer than 30 min.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
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.)
The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
To establish a culture from the frozen state, place an ampule in a 35°C water bath (2-3 min). Immerse the vial just sufficiently to cover the frozen material. Do not agitate the vial.
Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 10 mL complete medium in a T-25 flask.
Add 0.1 to 0.5 mL from a preparation of heat-killed Escherichia coli bacteria (ATCC® 23740™ or similar).
Incubate with the cap tightly sealed at 20-25°C.
Follow the protocol for maintenance of culture.
Name of Depositor
M Pelandakis
Chain of Custody
ATCC <-- M Pelandakis, Univ. Claude Bernard, Lyon, France
References
Atlan D, et al. Micriamoeba tesseris nov. gen. nov. sp.: a new taxon of free-living small-sized amoebae non-permissive to virulent Legionellae. Protist 163(6): 888-902, 2012. PubMed: 22677099
