Naegleria fowleri Carter (ATCC® 30214)

Strain Designations: Nf 66  /  Depositor: W Balamuth  /  Biosafety Level: 2

Strain Designations Nf 66
Biosafety Level 2

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.

10-year-old boy, South Australia, 1966
Product Format frozen
Type Strain no
Zymodeme Fw-1
Freeze preservation of pathogenic and nonpathogenic species
Medium ATCC® Medium 1034: Modified PYNFH medium (Available from ATCC as ATCC cat. no. 327-X)
Growth Conditions
Temperature: 30.0°C
Duration: axenic
1.  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.

2.  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.

3.   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.

4.  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 before the freezing process is begun should be no less than 15 min and no longer than 60 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 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 the ampules into liquid nitrogen.

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

8.   To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial enough to cover only the frozen material. Do not agitate the vial.

9.   Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate into 5.0 ml of fresh ATCC medium 1034.

10.          Incubate the tube on a 15° horizontal at 35°C with the cap screwed on tightly.

Name of Depositor W Balamuth
Chain of Custody
ATCC <<--W Balamuth<<--J.L. Griffin <<--- R.F. Carter
Year of Origin 1966

Carter RF. Primary amoebic meningo-encephalitis. An appraisal of present knowledge. Trans. R. Soc. Trop. Med. Hyg. 66: 193-213, 1972. PubMed: 4558822

Adams M, et al. A genetic approach to species criteria in the amoeba genus Naegleria using allozyme electrophoresis. Int. J. Parasitol. 19: 823-834, 1989. PubMed: 2635158

Simione FP Jr., Daggett PM. Freeze preservation of pathogenic and nonpathogenic Naegleria species. J. Parasitol. 62: 49, 1976. PubMed: 1255382

De Jonckheere JF. A century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 41: 309-342, 2002.