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1. Harvest cells from several cultures that are in the late logarithmic to early stationary phase of growth.  Place culture vessels on ice for 20-30 min.
2. Vigorously invert tubes 20-30 times or as necessary to sufficiently detach cells, then centrifuge at 500 x g for 5 min.  Handle cultures promptly after centrifugation to avoid the amoebae reattaching to the culture tubes.  Note:  Increased yield may be obtained by using a sterile cotton swab to rub the inside surface of culture tubes both before and after centrifugation.  Use of a refrigerated centrifuge will aid in preventing reattachment of cells to culture tubes during or immediately following centrifugation.        
3. Adjust the concentration of cells to between 5 x 10⁵/mL - 5 x 10⁶/mL using reduced medium (i.e., supernatant).
4. Mix the cell preparation and the cryoprotective solution in equal portions.  Invert the tube several times to obtain a uniform cell density.
5. Dispense 0.5 mL aliquots into 1.0 - 2.0 ml plastic sterile cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.  Use the following cooling cycle: From room temperature cool at -10°C/min to the heat of fusion; from the heat of fusion to -40°C, cool at -1°C/min.  At -40°C plunge into liquid nitrogen.  The cooling cycle should be initiated no less than 15 and no more than 30 minutes after the addition of DMSO to the cell preparation.
7. Store ampules in a liquid nitrogen refrigerator until needed.
8. To establish a culture from the frozen state, place an ampule in a 35°C water bath, until thawed (2-3 min).  Immerse the vial just sufficiently to cover the frozen material.  Do not agitate the ampule.
9. Aseptically transfer contents of thawed ampule to a glass, rubber-seal screw-capped tube containing 14-15 mL ATCC Medium 2832.
10. Screw cap on tightly and incubate on a 15° horizontal slant at 10-30°C (20-25°C recommended for routine cultivation).  Observe the culture daily and transfer when many trophozoites are observed.

Uchimura Y, et al. Mastigamoeba aflagellifera sp. nov. isolated from the soil of a rice field in Japan. (in preparation for submission)

Nucleotide (GenBank) : AB55091 ssu rRNA gene sequence of strain AF065-Y