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
Soil of a rice field, Kanagi, Goshogawara city, Aomori pref., Japan, Dec. 1, 2006
Product Format
frozen
Storage Conditions
Frozen Cultures: -70°C for 1 week; liquid N2 vapor for long term storage
Freeze-dried Cultures: 2-8°C
Live Cultures: See Protocols section for handling information
Type Strain
yes
Medium
ATCC® Medium 2832: Reduced YPD Medium
ATCC® Medium 2154: LYI Entamoeba medium
Growth Conditions
Temperature: 10°C to 30°C Atmosphere: Microaerophilic Culture System: Axenic
Cryopreservation
Reagents Cryoprotective Solution
DMSO, 2.0 mL
Fresh growth medium, 8.0 mL
Harvest and Preservation
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.
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.
Adjust the concentration of cells to between 5 x 105/mL - 5 x 106/mL using reduced medium (i.e., supernatant).
Mix the cell preparation and the cryoprotective solution in equal portions. Invert the tube several times to obtain a uniform cell density.
Dispense 0.5 mL aliquots into 1.0 - 2.0 ml plastic sterile cryules (special plastic vials for cryopreservation).
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.
Store ampules in a liquid nitrogen refrigerator until needed.
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.
Aseptically transfer contents of thawed ampule to a glass, rubber-seal screw-capped tube containing 14-15 mL ATCC Medium 2832.
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.
Name of Depositor
A Tonouchi
Chain of Custody
ATCC <-- A Tonouchi
References
Uchimura Y, et al. Mastigamoeba aflagellifera sp. nov. isolated from the soil of a rice field in Japan. (in preparation for submission)
