Acanthamoeba sp. (ATCC® 50497)

Strain Designations: BCM:88-2-37  /  Depositor: RJ Gast, TJ Byers  /  Biosafety Level: 2

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Strain Designations BCM:88-2-37
Biosafety Level 2
Isolation
human cornea, Houston, TX, 1988
Product Format frozen
Type Strain no
Comments
Subgenus systematics based upon SSU rDNA sequence data
Axenic
Medium Medium 712: PYG w/ Additives
Growth Conditions
Temperature: 25.0°C
Duration: axenic
Protocol: ATCCNO: 30135 SPEC: This strain is distributed as a frozen stabilate. See general instructions for thawing and storage of frozen material before proceeding. As soon as the shipment arrives, remove the frozen ampule from the dry ice and transfer it directly to a 35C water bath. After thawing the ampule, transfer the contents to a 16 x 125 mm plastic screw-capped test tube containing 5 ml of fresh medium. (Glass test tubes may also be used, but the cultures can be transferred less frequently when maintained in plastic.) Screw the cap on tightly and incubate the tube on a 5-15 degree slant at the appropriate temperature. Subculture every 2-4 weeks by vigorously agitating the culture and aseptically transferring a 0.1 ml aliquot to a fresh tube of medium. Prolongation of the transfer interval can be extended up to 6 months for certain strains of Acanthamoeba, however, this must be determined empirically for each strain.
Subcultivation
Protocol: ATCCNO: 30135 SPEC: This strain is distributed as a frozen stabilate. See general instructions for thawing and storage of frozen material before proceeding. As soon as the shipment arrives, remove the frozen ampule from the dry ice and transfer it directly to a 35C water bath. After thawing the ampule, transfer the contents to a 16 x 125 mm plastic screw-capped test tube containing 5 ml of fresh medium. (Glass test tubes may also be used, but the cultures can be transferred less frequently when maintained in plastic.) Screw the cap on tightly and incubate the tube on a 5-15 degree slant at the appropriate temperature. Subculture every 2-4 weeks by vigorously agitating the culture and aseptically transferring a 0.1 ml aliquot to a fresh tube of medium. Prolongation of the transfer interval can be extended up to 6 months for certain strains of Acanthamoeba, however, this must be determined empirically for each strain.
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 RJ Gast, TJ Byers
Special Collection NCRR Contract
Chain of Custody
ATCC <<--RJ Gast, TJ Byers<<--M.S. Osato strain Rawdon
Year of Origin 1988
References

Gast RJ, et al. Subgenus systematics of Acanthamoeba: Four nuclear 18S rDNA sequence types. J. Eukaryot. Microbiol. 43: 498-504, 1996. PubMed: 8976608

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

Ledee DR, et al. Advantages of using mitochondrial 16S rDNA sequences to classify clinical isolates of Acanthamoeba. Invest. Ophthalmol. Vis. Sci. 44: 1142-1149, 2003. PubMed: 12601042

Cross References

Nucleotide (GenBank) : U07410 nuclear SSU rRNA gene

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