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
Vero cell culture 120th passage, Bowling Green, OH, 1970
Product Format
frozen
Storage Conditions
Frozen: -80°C or colder Freeze-Dried: 2°C to 8°C Live Culture: See Propagation Section
Type Strain
no
Comments
Nutritional study of Naegleria gruberi
Medium
ATCC® Medium 1034: Modified PYNFH medium (Available from ATCC as ATCC cat. no. 327-X)
ATCC® Medium 935: NYTG Medium
ATCC® Medium 710: Nelson's Culture Medium For Naegleria
ATCC® Medium 803: M7 medium
ATCC® Medium 902: Schuster's axenic Naegleria medium
Growth Conditions
Temperature: 25°C
Culture System: Axenic
Cryopreservation
Harvest and Preservation
Harvest cells from a culture that is at or near peak density by centrifugation at 600 x g for 5 min. Pool the cell pellets into a single tube.
Adjust the concentration of cells to 2.0 x 106/mL. If the concentration is too low, centrifuge at 600 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
Prepare a 15% (v/v) sterile DMSO solution in ATCC medium 1034 as follows: Add the required volume of DMSO to a glass screw-capped test tube and place on ice. Allow the DMSO to solidify. Add the required volume of refrigerated ATCC medium 1034. Dissolve the DMSO by inverting several times. 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 106 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 60 min.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen.
The frozen preparations are stored in either the vapor or liquid phase of a nitrogen refrigerator.
To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just sufficiently to cover only the frozen material. Do not agitate the vial.
Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate into a fresh tube or flask of ATCC medium 1034.
Incubate at 25°C with the cap screwed on tightly (incubate a test tube on a 15° horizontal slant).
Name of Depositor
WD ODell
Year of Origin
1970
References
O'Dell WD, Brent MM. Nutritional study of three strains of Naegleria gruberi. J. Protozool. 21: 129-133, 1974. PubMed: 4206403
O'Dell WD, Stevens AR. Quantitative growth of Naegleria in axenic culture. Appl. Microbiol. 25: 621-627, 1973. PubMed: 4633479
De Jonckheere JF. A century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 41: 309-342, 2002.
