Trypanosoma gambiense Dutton (ATCC® 30025)

Strain Designations: Wellcome Ts  /  Depositor: EJ Tobie  /  Biosafety Level: 2

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Strain Designations Wellcome Ts
Application
Vector borne research
Biosafety Level 2
Isolation
human, Hospital for Tropical Diseases, London, England, 1921
Product Format frozen
Type Strain no
Growth Conditions
Duration: in vivo, laboratory rat
Protocol: ATCCNO: 30018 SPEC: This strain is distributed as a frozen stabilate. See general instructions for thawing and storage of frozen material before proceeding. Upon arrival remove the frozen ampoule from the dry ice and transfer directly to a 35C water bath. When completely thawed, aseptically remove the material with a syringe and inject 0.2 ml aliquots of the thawed contents of the ampule intraperitoneally into an adult rats known to be free of blood parasites. Draw a drop of blood from the tail of an infected rat. Add 1.0 ml of mammalian saline (0.8% saline buffered to pH 7.0) to the blood and examine a small amount microscopically. If one or more parasites are seen, proceed as follows: collect parasites into a syringe containing a buffered anticoagulant by either cardiac puncture or from the tail vein of the infected animal. Inoculate 0.2 ml of the diluted blood intraperitoneally into an adult rat known to be free of other blood parasites. Repeat the above procedures approximately every 4 days.
Subcultivation
Protocol: ATCCNO: 30018 SPEC: This strain is distributed as a frozen stabilate. See general instructions for thawing and storage of frozen material before proceeding. Upon arrival remove the frozen ampoule from the dry ice and transfer directly to a 35C water bath. When completely thawed, aseptically remove the material with a syringe and inject 0.2 ml aliquots of the thawed contents of the ampule intraperitoneally into an adult rats known to be free of blood parasites. Draw a drop of blood from the tail of an infected rat. Add 1.0 ml of mammalian saline (0.8% saline buffered to pH 7.0) to the blood and examine a small amount microscopically. If one or more parasites are seen, proceed as follows: collect parasites into a syringe containing a buffered anticoagulant by either cardiac puncture or from the tail vein of the infected animal. Inoculate 0.2 ml of the diluted blood intraperitoneally into an adult rat known to be free of other blood parasites. Repeat the above procedures approximately every 4 days.
Cryopreservation

Tyrode's Salt Solution

NaCl                                                                                    8.00 g

KCl                                                                                       0.20 g

CaCl2                                                                                   0.20 g

MgCl2 · H2O                                                                       0.05 g

NaH2PO4 · H2O                                                                  1.00 g

NaHCO3 · H2O                                                                   1.00 g

Glucose                                                                               1.00 g

Glass distilled H2O to                                                        1.00 L

Add ingredients in the sequence listed.  Filter-sterilize.

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 any 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% (v/v) DMSO in equal portions.  The final concentration will be 1.0 - 2.0 x 107 cells/ml and 5% DMSO.  The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process 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 rat.  Follow the protocol for maintenance in vivo.

Name of Depositor EJ Tobie
Chain of Custody
ATCC <<--EJ Tobie<<--C.A. Hoare
Year of Origin 1921
References

. . J. Parasitol. 36: 48-54, 1950.

Southworth GG, Read CP. Carbohydrate transport in Trypanosoma gambiense. J. Protozool. 16: 720-723, 1969. PubMed: 5362387

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