Cell Storage Procedure

Dicty Stock Center Cell Storage Procedure


  1. Harvest 1-2 x108 cells (1 clearing SM plate or axenic culture at appropriate density) for best viability results. 2 SM plates or 5 SM/5 plates for spores.

  2. Pre-cool foam storage container in -20°C freezer. Pre-cool 10, 1.5-ml screw-cap storage vials in an ice water tray.

  3. Pour 20 ml cold HL5 into sterile 100-ml beaker.

  4. With sterile spreader, scrape cells or fruiting bodies from plates and transfer to beaker with HL5.

  5. Pour the cells from the beaker into a 50-ml conical tube.

  6. Rinse the beaker with 10 ml HL5 to collect remaining cells and then pour that into the conical tube as well.

  7. Replace cap and shake tube.

  8. For axenic strains, just pipette desired amount into conical tube.

  9. Centrifuge cells at 200xg for 5 minutes. Centrifuge spores at 500xg for 5 minutes.

  10. Pour off supernatant.

  11. Resuspend pellet with 10 ml HL5 by vortexing (final density 1-2 x 107 cells per ml).

  12. Add 0.55 ml DMSO while gently swirling tube.

  13. Aliquot 1 ml of cell suspension into 10 storage vials.

  14. Replace caps, tighten well, then dry vials with paper towel before placing into pre-cooled foam container.

  15. Place foam container in -80°C freezer. Keep container away from metal to slow down the cooling rate.

  16. After 24 hours, vials can be transferred to liquid nitrogen.

After the vials have been in liquid nitrogen for several days, test the viability by scraping some cells from a vial (kept on dry ice) with a hot loop* and streaking them out on an SM/5 plate prespread with Klebsiella aerogenes. To test the viability and purity of an axenic strain, an entire vial should be thawed rapidly in a 37°C water bath and placed on ice as soon as the cell suspension has thawed. Do not allow cells to get too warm.

Pipette 50 µl of cells onto a plate with 0.25 ml K.a. and into 1 ml of HL5 in a 24-well plate. The remaining content of the vial can be transferred into a flask or a 100 x 20 mm style petri dish with 10 ml HL5.

* Even sturdy loops will bend out of shape, heat-kill some cells, and can melt the edge of storage tubes. A sturdy tool that gives much better plating results and no tube damage is Craftsman Professional No. 0 x 4 inch microtech Phillips head screwdriver, Sears # 00941527000 Mfr. Model # 41527. Heat the tip of the screwdriver and then cool in sterile water before using to scrape frozen cells from tube. Gently use tip of screwdriver to plate cells as usual.


  • Dictyostelium culturing procedures should take place in a sterile tissue culture hood. Plated cultures can be managed without it - though contaminants will be present. Axenic cultures, however, require sterile technique.

  • To prevent tubes from exploding, it is important to have the rubber o-ring stay in the cap during storage in liquid nitrogen. To keep o-rings from falling out, tighten caps all the way before initially opening vial.

  • If a vial is thawed, it should not be refrozen.

  • Viability dramatically decreases if cells are not immediately pipetted from the storage tube after thawing.

  • Storing cells at higher than suggested density decreases survivability.

  • We found that 5% DMSO gives better viabilities than 7.5% or 10% for our tested strains.

  • Storage in HL5 rather than horse serum is preferable if cells should ever need to be shipped internationally. There does not appear to be a difference in cell survivability between these two media.

S. Marsh. Last updated July 14, 2005