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How to handle frozen vials upon receipt

by Frédérique Foudrinier last modified 24-11-2015 12:27

 We recommend that the material be processed immediately upon arrival.

- Remove the frozen cryovial from the dry ice and transfer it rapidly to a 37oC water bath.

-   Homogenize by 2 reversals,

-   Wash to eliminate DMSO :

· Centrifuge 900 g for 10 minutes
· Discard supernatant
· Transfer the pellet in a sterile tube containing 6 mL of sterile physiologic
solution such as PBS buffer of 0,9% NaCl soultion (if inoculation to the
mouse) or 6 mL of culture medium (if cell culture).
. Centrifuge 900 g for 5 minutes and discard supernatant.

 

-   Treat immediately after :

· For cysts in brain tissue suspensions, it is better to inoculate them to Swiss mice (usually no more than 3 mice per aliquot).
· For tachyzoites, you can either reinoculate them to Swiss mice or add the entire thawed contents to a single small culture vessel (T-25 tissue culture flask, etc.) of the recommended medium and incubate at 37°C under 5% CO2.
 
If the material cannot be processed immediately, we recommend to store it in liquid nitrogen (temperatures below -130oC).
Frozen Toxoplasma  are stable in the long term only when stored at -130oC or below. Alternatively, the frozen material can be stored in a mechanical freezer between -70 and -80oC for short periods (1 to 5 days). Viability of parasites stored between -80 and -130oC will vary with the nature of the cryoprotective agent and the strain and can only be determined empirically.
 
One of the most common mistakes that users make in storing frozen material is to place it at -20oC (the freezer compartment of a conventional refrigerator-freezer). Never store frozen parasites at –20°C. Storage at this temperature for even short periods will result in certain death of the parasites.
 
 
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