Knowledgebase - Cryopreservation

Cryopreservation protocol from the Roscoff Culture Collection

Equipment

Chemicals and Solutions

Supplies

2 mL Cryogenic tubes (Simport, T311-2)
Brady Labels liquid nitrogen resistant (THT-181-492-3, ref VWR : BRDY805919)
Cryobox (Nalgène, 5026-0909)
Flasks with ventilation cap (Starlab CC7682-4825)
Aluminium foil

Cryopreservation protocol

Grow the strains to be cryopreserved until flasks are coloured.
Verify culture concentration daily by flow cytometry (or other method). Cryopreservation should be made 1 or 2 days after the beginning of the stationary phase.
Depending on the strain, add 5 or 10 % DMSO (Diméthylsulfoxyde) final concentration to 1mL of culture and transfer to a 2mL cryogenic tube.
Start the Progressive Freezer and run the following program :
1. Start at 20°C
2. Decrease temperature by 1°C per minute until -40°C
3. Hold 10 min at -40°C
When the cycle is finished, protect yourself with gloves, glasses, and lab coat. Plunge the cryogenic tubes into liquid nitrogen.
Place cryogenic tubes in cryoboxes and store in a -150°C freezer and/or in a liquid nitrogen container for long-term storage. In general we store 9 tubes in the freezer and 3 tubes in liquid nitrogen.

Culture Recovery

Quickly thaw cryogenic tube in a 25-30ºC water bath.
As soon as ice in the tube has melted, quickly clean the outside of the tube with 70% ethanol and transfer the cells into a flask containing 20mL fresh culture medium.
Surround the flasks with aluminum foil to keep them in the dark and put them at the optimal temperature for the culture.
After 24 hours, remove the aluminum foil.
Observe the cultures every other day (by colour and/or by flow cytometry and/or by microscopy).
Culture recovery can take a long time (1-2 months or even more).

This protocol was developed by Roseline Edern and Priscillia Gourvil at the Roscoff Culture Collection - version 3.0 July 2014.

Genera tested successfully