Cryopreserving Protists ‐ BEAP Lab - IBEMicrobeHub/culturing GitHub Wiki

Cryopreserving Protists - BEAP Lab

Materials:

  • Your sample in good condition.
  • Cryovials 1 mL (Thermo Fisher Nunc ref. 10529881).
  • Tweezers.
  • Needle nose pliers.
  • DMSO (the one not for PCR) or Glycerol.
  • Cut yellow tips if you use the glycerol because its density.
  • Nunc Cryoflex tubing (plastic protection, Thermo Fisher ref. 343958).
  • Flame + lighter.
  • Scissors
  • Mr Frosty - filled up to 250 mL with clean Isopropanol.
  • Special stickers for Liquid Nitrogen (LN2).
  • Security measures: lab coat, glasses and cryogenic gloves.

Method:

Always working under the biological hood.

A. Prepare the samples to freeze:

  1. Grow the culture in its medium (1-2 weeks. It is up on sample!) by doing the split (17mL of medium + 3mL of culture). Use an adequate volume, ~20mL, to freeze 10 cryovials for each sample.
  2. Label all 10 vials by putting Beap ID and day/month/year. NB: by reporting only the BEAP ID means that you have the information of the sample (media dilution, growth condition + other details) in this table: https://docs.google.com/spreadsheets/d/1xf8NksF57IzfZ9YdE2D0sstoiUwfC-5GLXE3JuY8d8o/edit#gid=736969780 .
  3. Cut the plastic protection in pieces of ~8 cm each.
  4. Add 900uL of sample in each cryovial.
  5. Then add 100 µL of DMSO (10% final concentration).

From this step to its storage into Mr frosty at -80ºC, don't waste time! Proceed rapidly because of the effect of the DMSO on the cells!

Note: alternatively DMSO 5% and 2,5%, OR Glycerol 10%, 5%, 2,5% as final concentration. Check or test which final concentration might work for your species/culture.

  1. Mix and then insert each prepared cryovial inside a cut cryogenic plastic tube.

Note: skip this step if you want to store the samples at -80ºC and move directly to (A.11).

  1. By using a tweezer, pass the cryovial on the flame until the plastic tube will be perfectly adherent!

Without gloves - BUT cleaned hands!

  1. Use a needle nose pliers to close the extremities properly.

This step is very important!

  1. Cut the excess of plastic with the scissors.

If you don’t do that, the cryobox cannot be closed.

  1. Repeat these last two passages for all your 10 cryovials.
  2. Move the vials to the cooling chamber Mr Frosty.
  3. Store Mr Frosty at -80ºC overnight.

At this point decide whether to store your sample:

B. Freezing at -80ºC:

  1. The day after, move your vials in the box nºX at -80ºC.
  2. Update the table cultures at -80ºC: https://docs.google.com/spreadsheets/d/1xf8NksF57IzfZ9YdE2D0sstoiUwfC-5GLXE3JuY8d8o/edit#gid=1231327303 .

C. Recovery the samples from -80ºC:

  1. Prepare the biological hood with all the materials.
  2. Fill a flask with 10mL fresh growing medium.
  3. Take out the frozen stock from the freezer, carry it to the culture hood in a cryo box or ice to prevent it from thawing.
  4. By using a loop move the content and resuspend it in the culture flask.

Note: use the loop only if the content inside the cryovial is rigid. Otherwise, if liquid, move the content by using a gilson.

  1. Let it recover for at least 3-4 days (or a week, it is up to the culture) and proceed with another transfer in a fresh medium even if the cells are not completely grown. It will help to dilute the remaining DMSO.

Note: because of DMSO, some precipitates may appear in the culture.

D. Move the samples from -80ºC to the Liquid Nitrogen dewar:

Security measures: lab coat, LN2 gloves, glasses and tweezers.

  1. Move and clean the lab footstool from the X3 room to a space closed to the LN2 dewar.

The footstool will be used to put the frozen rack.

  1. By applying the security measures reported above, open the LN2 dewar and take out the rack of your interest. Take the rack out very slowly because the LN2 will drop in the meantime.

From this point to on, you must do everything within 3-4 minutes!(Why? because you are working at RT and the rack - containing your samples - MUST NOT unfreeze!)

  1. Take the cryobox of your interest.
  2. Move your cryovials from Mr Frosty to the cryobox.
  3. Put the cryobox back into the rack.
  4. Put the rack back into the LN2 dewar and close the cap!
  5. Update the table: https://docs.google.com/spreadsheets/d/1xf8NksF57IzfZ9YdE2D0sstoiUwfC-5GLXE3JuY8d8o/edit#gid=736969780 .

E. Recovery the samples from Liquid Nitrogen:

  1. Prepare the biological hood with all the materials.
  2. Fill a flask with 10mL fresh growing medium.
  3. Take out the frozen stock from the LN2, carry it to the culture hood in a cryo box or ice to prevent it from thawing.
  4. Cut the plastic protection.
  5. By using a loop move the content and resuspend it in the culture flask.

Note: use the loop only if the content inside the cryovial is rigid. Otherwise, if liquid, move the content by using a gilson.

  1. Let it recover for at least 3-4 days (or a week, it is up to the culture) and proceed with another transfer in a fresh medium even if the cells are not completely grown. It will help to dilute the remaining DMSO.

Note: because of DMSO, some precipitates may appear in the culture.

Further information

How do you test liquid nitrogen levels?

To use, place a measuring stick to the bottom of the liquid nitrogen dewar, hold for 4-5 seconds, remove, and allow frost to form on the measuring stick. A frost line will appear at the height of the liquid nitrogen on the measuring stick. The actual level may be up to 1/2 inch lower due to splashing from the liquid nitrogen.

Care and Maintenance of a Liquid Nitrogen Tank

https://www.youtube.com/watch?v=muAQEB7Fcvo