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
thermally polluted canal, Belgium, 1974
Product Format
frozen
Storage Conditions
Frozen: -70°C or colder for 1 week, vapor phase of liquid nitrogen for long-term storage
Axenic/Xenic
Xenic, grown with mixed bacteria
Type Strain
no
Comments
Comparative study of Naegleria strains
Axenic medium for differentiation of pathogenic species
isoenzyme patterns of pathogenic and non-pathogenic spp.
Isoenzyme identification of pathogenic species
Medium
ATCC® Medium 997: Fresh water ameba medium
Growth Conditions
Temperature: 35°C
Cryopreservation
Allow the cells to encyst. To detach cysts from the plate flush the surface with 5 ml fresh ATCCâ medium 1323 (Page's Balanced Salt Solution). Rub the surface of the plate with a spread bar to detach adhering amoebae.
Transfer the cyst suspension to a sterile centrifuge tube.
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.
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.
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 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 freezer.
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.
Immediately after thawing, aseptically remove the contents of the ampule and distribute to the center of a fresh plate of ATCC medium 997. Distribute the material evenly over the plate using a spread bar. Incubate at 35°C.
Name of Depositor
JF De Jonckheere
Year of Origin
1974
References
De Jonckheere J. Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates. Appl. Environ. Microbiol. 33: 751-757, 1977. PubMed: 869525
De Jonckheere J, van de Voorde H. Comparative study of six strains of Naegleria with special reference to nonpathogenic variants of Naegleria fowleri. J. Protozool. 24: 304-309, 1977. PubMed: 881654
De Jonckheere JF. Isoenzyme patterns of pathogenic and non-pathogenic Naegleria spp. using agarose isoelectric focusing. Ann. Microbiol. 133: 319-342, 1982.
De Jonckheere JF. Isoenzyme patterns of pathogenic and non-pathogenic Naegleria spp. using agarose isoelectric focusing. Ann. Microbiol. (Paris) 133: 319-342, 1982. PubMed: 6211120
De Jonckheere JF. A century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 41: 309-342, 2002.
