Trypanosoma lewisi (Kent) Laveran and Mesnil (ATCC® 30211)

Organism: Trypanosoma lewisi (Kent) Laveran and Mesnil  /  Depositor: CM Lee

Permits and Restrictions

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Strain Designations Sp
Application
Vector borne research
Biosafety Level 1
Isolation
feral rat, Rattus sp., Singapore, 1962
Product Format frozen
Type Strain no
Growth Conditions
Duration: in vivo, laboratory rat
Protocol: ATCCNO: 30085 SPEC: Inject the entire thawed contents of the ampule intraperitoneally into an adult rat known to be free of blood parasites. The strain should be monitored, especially after infecting with thawed material. To infect additional rats, draw a drop of tail blood into a 1.0 ml tuberculin syringe containing 0.5 ml of sterile buffered anticoagulant, mix, and inoculate 0.1-0.2 ml of the suspension intraperitoneally/rat. Only a few trypanosomes are needed to infect a non-immune rat. However, do not wait until the blood is swarming with trypanosomes. The time for peak parasitemia may be within 4-5 days if large inocula are given. Peak parasitemia from the thawed material may be slightly sooner or later.
Subcultivation
Protocol: ATCCNO: 30085 SPEC: Inject the entire thawed contents of the ampule intraperitoneally into an adult rat known to be free of blood parasites. The strain should be monitored, especially after infecting with thawed material. To infect additional rats, draw a drop of tail blood into a 1.0 ml tuberculin syringe containing 0.5 ml of sterile buffered anticoagulant, mix, and inoculate 0.1-0.2 ml of the suspension intraperitoneally/rat. Only a few trypanosomes are needed to infect a non-immune rat. However, do not wait until the blood is swarming with trypanosomes. The time for peak parasitemia may be within 4-5 days if large inocula are given. Peak parasitemia from the thawed material may be slightly sooner or later.
Cryopreservation

1.   Harvest the parasites according to the protocol for maintenance in vivo.

2.   Spin the cell suspension at approximately 50 x g for 3 min, to remove the cellular debris.

3.   Transfer the supernatant to a new 15 ml plastic centrifuge tube.  Centrifuge at 1300 x g for 10 min.

4.   Pool the cell pellets and adjust the concentration to 2.0 - 4.0 x 107 cells/ml with a fresh solution of Tyrode's Salt Solution.

      *If the concentration is too low centrifuge at 1300 x g for 10 min and resuspend in the volume of Tyrode's Salt Solution required to yield the desired concentration.

5.   Mix the cell preparation and 10% DMSO (v/v) Tyrode's Salt Solution in equal portions.  The final concentration will be 1.0 - 2.0 x 107 cells/ml and 5% DMSO in Tyrode's Salt Solution.  The time from the mixing of the cell preparation and the cryoprotective solution before the freezing process begins should be no less than 15 min. and no more than 30 min.

6.   Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

7.   Place the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at        -1°C/min through the heat of fusion.  At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.  Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.  (The cooling rate in this apparatus is approximately

-1°C/min.)  

8.   Store in either the vapor or liquid phase of a nitrogen refrigerator.

9.   To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.  Do not leave ampule in water bath after thawed.

10. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected mouse.  Follow the protocol for maintenance in vivo.

Name of Depositor CM Lee
Chain of Custody
ATCC <<--CM Lee<<--D. Lincicome
Year of Origin 1962
References

. . Exp. Parasitol. 23: 114-127, 1968.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation