Cryopreserving Protists ‐ MCG Lab - IBEMicrobeHub/culturing GitHub Wiki

Cryopreserving in 10% DMSO

This procedure works for Acanthamoeba castellanii, Amoebidium parasiticum, Dictyostelium discoideum, Chromosphaera perkinsii (NK52), Pirum gemmata, Sphaeroforma arctica, Spizellomyces, Capsaspora owczarzaki, Corallochytrium and Creolimax fragantissima.

Material:

For freezing the cells (part A).

  • DMSO
  • Screw-top cryovials
  • Cells
  • Mr. Frosty unit. It is located in the main lab. Make sure it is filled with clean isopropanol before using it.

For recovery the cells (part B).

  • Fresh medium
  • Flaks
  • Cryobox

Method:

A) Freezing

  1. Pre-grow the culture in appropriate growing medium until almost saturated but not too old.

  2. Mix 1.35mL of the culture with 150uL of sterile DMSO (1.5mL final volume) in the screw-top cryovials.

  3. Transfer the cryovials to the cooling chamber Mr Frosty unit.

  4. Place the Mr Frosty unit in the -80ºC freezer. Leave it overnight.

  5. Remove cryovials from Mr. Frosty unit and store in appropriate boxes for -80ºC.

B) Recovery

  1. Fill a flask with 10mL fresh growing Medium.

  2. Take out the frozen stock from the freezer, carry it to the culture hood in a cryo box to prevent it from thawing.

  3. With a pipette tip (or a loop), take a chunk of the frozen stock and resuspend it in the culture flask (~10uL is probably sufficient). Take the frozen stock back to the -80ºC as rapidly as possible.

  4. Let it recover for at least 3-4 days 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 from the cryopreservation, the time will vary depending on the amount of the initial inoculum and the culture type.

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

All protists must be stored in liquid nitrogen for long-term storage. Although storage at -80°C is sometimes sufficient for a temporary alternative.

Cryopreserving in 5% DMSO

This procedure works for Naegleria gruberi and Abeoforma whisleri.

Material:

  • DMSO
  • Screw-top cryovials
  • Cells
  • Mr. Frosty unit. It is located in the main lab. Make sure it is filled with clean isopropanol before using it.

Method:

  1. Prepare 10% v/v DMSO in growth medium:
  • Place the cryovial on ice, let it refrigerate.
  • Add 75uL of DMSO.
  • Add 675 uL of refrigerated medium.

  1. Mix to dissolve the DMSO by inverting the tube several times. Place it on ice.

  2. Add 750uL of confluent culture. Mix by inverting.

  3. Transfer the cryovials to the cooling chamber Mr Frosty unit.

  4. Place the Mr Frosty unit in the -80ºC freezer. Leave it overnight.

  5. Remove cryovials from Mr. Frosty unit and store in appropriate boxes for -80ºC.

Cryopreserving in 10% of DMSO for Abeoforma whisleri

10%DMSO in 1M sorbitol or 10%(glycerol 60%) in sorbitol 1M (no culture medium).