Naegleria fowleri Carter (ATCC® 30175)

Organism: Naegleria fowleri Carter  /  Depositor: CG Culbertson

Permits and Restrictions

View Permits

Strain Designations HB2
Biosafety Level 2
Isolation
human, Orlando, FL, 1968
Product Format frozen
Type Strain no
Comments
Can be cultivated in ATCC medium 1034 without the cell line.
Medium ATCC® Medium 1034: Modified PYNFH medium (Available from ATCC as ATCC cat. no. 327-X)
ATCC® Medium 1034: Modified PYNFH medium (Available from ATCC as ATCC cat. no. 327-X)
ATCC® Medium 722: Minimum essential medium (MEM)
Growth Conditions
Temperature: 35.0°C
Duration: grown with Macaca mulatta kidney cells, ATCC CCL 7.1
Protocol: ATCCNO: 30100 SPEC: Grown with Macaca mulatta kidney cells, ATCC CCL 7.1. Kidney cells for use with the amebae are grown in screw-capped tubes containing 2.0 ml of ATCC medium 722. When tissue cultures are well established, inoculate with 0.1 ml from a flourishing amebae parent culture. Cultures must be gassed with 10% CO2 and 90% air after inoculation and at 4-day intervals. Incubate inoculated amebae cultures on a slant.
Subcultivation
Protocol: ATCCNO: 30100 SPEC: Grown with Macaca mulatta kidney cells, ATCC CCL 7.1. Kidney cells for use with the amebae are grown in screw-capped tubes containing 2.0 ml of ATCC medium 722. When tissue cultures are well established, inoculate with 0.1 ml from a flourishing amebae parent culture. Cultures must be gassed with 10% CO2 and 90% air after inoculation and at 4-day intervals. Incubate inoculated amebae cultures on a slant.
Cryopreservation
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 CG Culbertson
Year of Origin 1968
References

Butt CG, et al. Naegleria (sp.) identified in amebic encephalitis. Am. J. Clin. Pathol. 50: 568-574, 1968. PubMed: 5748971

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

Basic Documentation