Leishmania donovani (Laveran and Mesnil) Ross (ATCC® 30505)

Organism: Leishmania donovani (Laveran and Mesnil) Ross  /  Depositor: R Herman

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Deposited As Sclerotium bataticola Taubenhaus, anamorph
Strain Designations GM [Mediterranean Strain - Line #1]
Application
Vector borne research
Biosafety Level 2
Isolation
human bone marrow (patient infected in Greece), Johns Hopkins Hosp., Baltimore, MD, 1968
Product Format frozen
Type Strain no
Comments
Amastigotes.
Growth Conditions
In vivo, Golden hamster
Subcultivation
Protocol: ATCCNO: 11745 SPEC: See general instructions for thawing and storage of frozen material before proceeding. Add thawed contents to a single 16 x 125 mm glass screw-capped test tube of the appropriate medium. Incubate the culture vertically with the cap screwed on tightly. It is essential to establish cultures initially in small volumes. Once established, the culture can be scaled up to larger volumes. Vigorously agitate the culture and aseptically transfer 0.1 ml of culture to a fresh tube of medium weekly.
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 from spleen tissue homogenized in a balanced salt solution (i.e., Tyrodes' Salt soln. or similar), approximately 5.0 ml solution per spleen.

2.   Transfer the cell homogenate to a 15 ml plastic centrifuge tube and spin at approximately 1300 x g for 10 min.

3.   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.

4.   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.

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

6.   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.)  

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

8.   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.

9.   Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected Golden hamster at least 8 weeks old.  Follow the protocol for maintenance in vivo.

Name of Depositor R Herman
Chain of Custody
ATCC <<--R Herman<<--L. Stauber <<--- E.L. Schiller
Year of Origin 1968
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