Diplomonads:Freeze Giardia

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To Freeze Giardia in cryovials

1. Prepare a separate solution of sterile filtered 18% DMSO in culture medium (e.g., take 4.1 ml medium, add 0.9 ml DMSO and sterile filter using a syringe filter).
2. Label liquid nitrogen vials with name of cell line, strain of Giardia, date and your initials. Record this in culture book.
3. Ice cells for 15 minutes.
4. Spin down 12mL of confluent culture for 5 min at (900 x g) @4 degrees Celsius.
5. Pour off all supernatant and add 1ml of fresh culture medium.
6. Add 1ml 18% DMSO to 1ml resuspended culture for a final [9%]. Mix gently and aliquot into two liquid nitrogen vials using a sterile transfer pipet
7. Transfer to –80°C for several days. Move to liquid nitrogen tank for permanent storage.

To thaw Giardia from cryovial

1. Bring stored vial rapidly to 37°C. Record removal of frozen aliquot in the culture book.
2. Pour into a 15ml culture tube with fresh medium, and let attach at 37°C for about 60 minutes.
3. Pour off supernatant and replace with fresh medium (no DMSO). Add puromycin at this point, if necessary.
4. Culture will be confluent the following day.