Cryopreservation protocol from the Roscoff Culture Collection
- Flow cytometer (to measure cell concentration)
- Laminar flow hood
- Brady Printer
- Planer Progressive Freezer (Kryo 360-3.3)
- Isothermal box (for liquid nitrogen during transport)
- Liquid nitrogen tank
- -150°C freezer
- Water bath
- Gloves, protective glasses and lab coat
Chemicals and Solutions
- Culture Medium
- Dimethylsulfoxyde (DMSO)
- Liquid nitrogen,
- 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 (Sarstedt, 83.1810.002)
- Aluminium foil
- 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 :
- Start at 20°C
- Decrease temperature by 1°C per minute until -40°C
- 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.
- 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
- Clade VII