Acanthamoeba stevensoni Sawyer et al. (ATCC® 50388)

Strain Designations: RB-F-1  /  Depositor: TK Sawyer  /  Biosafety Level: 2

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Strain Designations RB-F-1
Biosafety Level 2
Isolation
sewage-contaminated hard clam, Mercenaria mercenaria, beds in Raritan Bay, NY, near the Staten Island shoreline
Product Format freeze-dried
Type Strain yes
Comments
species description
Medium Medium 997: Fresh water ameba medium
Growth Conditions
Temperature: 25.0°C
Max Temperature: 35.0°C
Min Temperature: 25.0°C
Protocol: ATCCNO: 30011 SPEC: This strain is distributed as a dried preparation. See the general procedures for opening a dried vial. Aseptically add 1 ml of sterile distilled water to the inner shell vial, remove the filter paper aseptically with a pair of forceps, and place it in the center of an agar plate of ATCC medium 997. Add the liquid remaining in the vial to the plate and spread it evenly over the surface of the plate. Incubate the plate at 25C. Trophozoites (amebae) should be evident within 2-3 days.
Subcultivation
Protocol: ATCCNO: 30011 SPEC: This strain is distributed as a dried preparation. See the general procedures for opening a dried vial. Aseptically add 1 ml of sterile distilled water to the inner shell vial, remove the filter paper aseptically with a pair of forceps, and place it in the center of an agar plate of ATCC medium 997. Add the liquid remaining in the vial to the plate and spread it evenly over the surface of the plate. Incubate the plate at 25C. Trophozoites (amebae) should be evident within 2-3 days.
Cryopreservation

1.  Allow the cells to encyst.  To detach cysts from the plate flush the surface with 5 ml fresh ATCC medium 1323 (Page's Balanced Salt Solution).  Rub the surface of the plate with a spread bar to detach adhering cysts.

2.   Transfer the liquid medium to a sterile centrifuge tube.

3.  If the cyst concentration does not exceed 2 x 106 cysts/ml adjust the suspension to that concentration.  To adjust the concentration, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield 2 x 106.

4.  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.

5.  Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be at least 106 cysts/ml and 7.5% (v/v) DMSO.  The equilibration time (the time between addition of DMSO and the start of the cooling cycle) should be no less than 15 min and no longer than 30 min.

6.   Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

7.   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.)  

8.  The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.

9.   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 to a level just above the surface of the frozen material. Do not agitate the vial.

10.          Immediately after thawing, aseptically remove the contents of the ampule and distribute to the center of a fresh plate of ATCC medium 711.  Distribute the material evenly over the plate using a spread bar.  Incubate at 25°C.

Name of Depositor TK Sawyer
Special Collection NSF - Protistology
References

Sawyer TK, et al. Acanthamoeba stevensoni n. sp. (protozoa: amoebida) from sewage-contaminated shellfish beds in Raritan Bay, New York. J. Eukaryot. Microbiol. 40: 742-746, 1993.

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

Kong HH, et al. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea. J. Clin. Microbiol. 40: 1199-1206, 2002. PubMed: 11923331

type strain

Cross References

Nucleotide (GenBank) : AF019069 Acanthamoeba stevensoni RB:F:1 18S ribosomal RNA gene, partial sequence.

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