Naegleria gruberi Schardinger

Establishing Cultures from Dried State

This strain is grown with Echerichia coli and then shipped as a dried preparation.

1. To rehydrate an ampule, aseptically add 1 ml of sterile distilled water to the inner shell vial.  Aseptically remove the filter paper pellet with a pair of forceps, and place it in the center of a plate of ATCC medium 997.
2. Add the liquid remaining in the vial to the plate, tease apart the filter paper pellet, and distribute evenly over the surface of the plate.
3. Incubate the plate upright at 25°C.  Trophozoites should be seen within 2-3 d.

1. Remove an agar block (~5 mm² ), with trophozoites or cysts, from the edge of an agar plate culture and place it in a test tube containing 1 ml of sterile ATCC medium 1325.  Agitate to suspend cells from the agar block.  Transfer 0.25 ml of the solution to center of each of two fresh plates and spread evenly with a spread bar.
2. Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.
3. Repeat steps 1-3 at 10-14 d intervals.

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 amoebae.
2. Transfer the cyst suspension 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 60 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 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.
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 997.  Distribute the material evenly over the plate using a spread bar.  Incubate at 25°C.