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
Human, Nantucket Island, MA, 1970
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
no
Comments
Infection with piroplasms isolated from man
Growth Conditions
Culture System: in vivo, hamster
Cryopreservation
Reagents Alsever's Solution
NaCl, 4.2 g
Na3citrate•2H2O, 8.0 g
Glucose, 20.5 g
Glass distilled H2O to 1.0 L
*Dissolve components in glass distilled H2O, adjust the pH to 6.1 with 10% (w/v) citric acid and filter sterilize. The solution can be obtained from Sigma-Aldrich (cat# A3551).
Harvest and Preservation
Prepare a 30% (v/v) sterile glycerol solution in Alsever's solution.
Draw approximately 0.5 mL of anticoagulant solution (Yaeger's or heparin, etc.) into a syringe and move it back and forth over the length of the syringe, several times. Remove all air bubbles. Draw blood by cardiac puncture into the syringe from a host animal that has reached or is near peak parasitemia. If clotting occurs during extraction of blood, insufficient heparin was used.
Mix the heparinized blood with the 30% glycerol solution in a 2:1 ratio. If any clotting has occurred do not use. After mixing, the final concentration of cryoprotectant solution will be 10% (v/v). The mixture should be placed in a 4°C ice bath. The time from the mixing of the cell preparation and glycerol stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation). Filled ampules should be placed in a 4°C ice bath. Do not immerse ampules to the level of the vial cap.
Plunge ampules from 4°C into liquid nitrogen. The frozen preparations may be stored in a mechanical freezer until needed, however storage in either the vapor or liquid phase of a nitrogen refrigerator is recommended for the longest viability.
To thaw a frozen ampule, place in a 35°C water bath, until thawed (2-3 min). Immerse the ampule just sufficient to cover the frozen material. Do not agitate the ampule.
Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected hamster. Follow the protocol for maintenance in vivo. The course of infection may be longer or shorter than usual depending on percent recovery of the parasite from the frozen state.
Name of Depositor
JP Kreier
Chain of Custody
ATCC <-- JP Kreier <-- A. Holbrook <--- . . . <-- K.A. Western
Year of Origin
1970
References
Van Peenen PF, Healy GR. Infection of Microtus ochrogaster with piroplasms isolated from man. J. Parasitol. 56: 1029-1031, 1970. PubMed: 5504524
