Dear All, We are faced with preserving hundreds of clones we have picked up from wild samples and are trying to decide what is the best way to do it. We are aware of methods for lyophilizing samples but this is rather tedious. We plan to use it only for our most valuable samples. We could save spores in silica, but it is unclear how long this will last. Any thoughts on this or recent protocol modifications would be welcome. It seems like most people freeze cells in HL5 with 10% DMSO. Does this last months or years? Is it necessary to centrifuge the samples and collect the pellet and resuspend before freezing? Is there a protocol for freezing spores that people have had success with? In principle it seems like spores should be easier to save than cells, but maybe freezing won't work well with them. Any opinions would be welcome.

Joan E. Strassmann, Professor
Dept. of Ecology and Evolutionary Biology, MS 170
Rice University

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Dear Dicty Listserv,
Here's what I've learned from all you helpful folks. First I give my synthesis and below I append the various emails I received so you can come to your own conclusions. Please let me know if I've made an error. First, save spores not cells if possible. Its easier and more reliable. It lets nature do the work of getting the clones into a preservable form. To do this you bang spores into a petri dish lid, wash them into a cryovial and freeze at -80. These can keep for many years. What you use to wash them into the cryovial is variable. If there really isn't any water left inside the spores, about anything should work. People reported success with phosphate buffer, HL5, milk+glycerol and other combinations. If there is some water lurking in there, maybe quick freezing in liquid nitrogen or a frosty freeze and glycerol in the solution will help. However, I can't see how glycerol could get into the spore to protect it from intracellular ice crystals. Maybe the protein in milk or HL5 offers some extra coating. We plan to use SM and freezing for our clones which all make spores. Soon we'll set up a test of the various methods. If you must save cells a few precautions can make a big difference. First of all, DMSO needs to be fresh and high quality (see detailed email below). The cells need to be highly concentrated and clean. The emails below give the details. Cells emails are first, then spore emails. Thanks again to everyone.

Joan

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CELLS

From: "Elizabeth J. Luna" Elizabeth.Luna@UMassmed.edu
Hi Joan,
Two factors that we have found to be important for getting reasonably long-term viability from cells frozen in HL5 with 10% DMSO:
1. The DMSO must be tissu e-culture grade and stored under nitrogen. After exposure to oxygen for prolonged periods, this solvent oxidizes to products that can kill the cells. You want to use tissue-culture grade DMSO from a freshly-broken vial.
2. The cells must be concentrated ("5 x 10^8 cells/ml). This means that you will need to centrifuge axenically-grown cells, which plateau at 10^7 cells/ml. For your wild samples, you also will want to wash away most bacteria before resuspending in a minimal amount of HL5. Dilute suspensions of frozen cells usually die off within weeks to months; concentrated suspensions are good for 1-5 years. For all our really important stocks, we re-make the frozen stocks at least once per year.
It would be great if you would let us all know if anyone has better techniques.
Good luck,

Beth Luna
Elizabeth J. Luna, Ph.D.
Professor of Cell Biology
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Hi,
I have used 7.5% DMSO in HL5 media to store axenic strains for >5 years with no apparent loss in viability. The main problem with this method is the potential freezer failure. You should note that DMSO is reasonably nasty stuff; it is important to get the cells into the freezer shortly after addition of the freezing medium. BUT DO NOT snap freeze them. Just stick the cells in the -80C and they will be fine. Also, it has been my experience that changing the media just prior to freezing is necessary for successful recovery of strains. Perhaps you could place the cells in 96 well microtiter plates Another option might be the plates for bacterial cell culture used by people doing high-throughput genomics (?). Some centrifuges are equipped to allow you to spin samples in this kind of format. This would enable you to rapidly and easily exchange the medi a prior to freezing. In addition I believe there are ways to seal these plates can be sealed and stored at -80C
Good Luck!

Anne L. Hitt
** We store cells at -70 suspended in a mixture of HL-5, DMSO, and fetal bovine serum that lasts for at least 6 years so far.
Solution A: 0.42 ml fetal calf serum, 1.25 ml HL-5
Solution B: 0.56 ml fetal calf serum, 1.25 ml HL-5, 0.45 ml DMSO

Filter sterilize and store at -20
1. Harvest 4 x 10^7 cells and resuspend in solution A (1.7 ml)
2. Add 2.3 ml solution B dropwise while mixing the cells by shaking the tube. total volume = 4 ml
3. Place 1 ml of cells in solution in a cryovial
4. Freeze in an isoproanol bath at -80

To thaw:
Let cells thaw at room temp.
Spin down the cells in the cryovial and aspirate the DMSO containing media.
Resuspend in a small volume of HL-5
Add cells to a T-25 flask with about 6-8 ml in it.
Let them grow up overnight. (typically, there is a monolayer by the next morning). If the cells are mutants requiring selective drugs (such as G418 or blasticidin), add these drugs a day after the cells are pulled from the freezer.


SPORES

Freezing of spores works well and we do it routinely. Simply collect fruiting bodies in Soerensen Phosphate buffer, vortex and freeze in liquid nitrogen. Store at -70°C.
All the best,
Ralph
Dr. Ralph Graef

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Simply collect spores with a platinum loop and suspend in sterile 50%-100% glycerol. After keeping at -70 C for more than five years, they germinate actively when put into HL5 medium. I believe storage at -20 C should (and in a refregerator, maybe) also work fine. Old literatures document the effect of proteose peptone to facilitate germination, so I think HL-5 is better than buffer.
Good luck!
Yoshio
(From) Dr. Yoshio Fukui

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Dear Joan, Valuable species we lyophilize as well. Routinely we freeze spores as follows. Slam a plate with fruiting bodies right side up to the underside of a table. The majority of spores are now in the lid. Add 5ml of HL5 to the lid, pipette a few times up and down to resuspend all the spores and freeze in as many aliquots as yo u see fit at -80oC. These spores last for at least 10 years and they also survived a little mistake of D HL who sent all my cells/spores packed on dry ice by road transport to Italy instead of by air to Dundee when I moved last year. After 10 days at ambient temperature in summer in Italy and refreezing at -80oC, they still come up fine in Dundee. However, I would not recommend this as a routine procedure and it did not work for the frozen cells. Good luck, Pauline

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Dear Joan
A good way to preserve spores is to collect them in buffer (almost any kind will do but we have successfully used 10mM Kphosphate, pH6.6), 20% glycerol. I have stored them at minus 80 degrees for years with no loss of viability. The nice thing about this method is that if you want to grow up the culture you can thaw the frozen spores either a little bit or completely and take a sample for growth. I also think freezing at minus 20 degrees is okay but I do not have too much experience with this. Of course, freezing in liquid nitrogen is also okay.
Herb Ennis

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Hi,
You've gotten a few comments already on storing your strains. I think that frozen spores at -70 C is much more preferable than cells at -70 C. It's good with small numbers of spores. I have had good success with wild isolates stored on silica gel. Basically collect the spores as Pauline recommended by banging them onto the lid of a petri dish, resuspend them in about half an ml of serum and put them onto about five grams of coarse silica gel crystals in a screw top vial. Shake well or vortex to distribute the spores. Seal with parafilm and store a 4 C. The spores stored this way last about a decade sometimes longer.
Dennis L. Welker

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Dear Joan- I have been too busy to respond, but let me add a few points. First, we have never used specially stored DMSO, so although that might be a good precaution, I doubt it is necessary. We switched from phosphate buffered HL5 to Bis-Tris HL5 at the recommendation of a tissue culture lab who claimed that you can get some kind of funny pH effects on long term storage of phosphate buffered solutions so that you get local pH increases and decreases. Since we switched, we believe our stocks last much longer, but we have no empirical data. For freezing DMSO's we set up the tubes in an ice bucket change the media in the plate to Bis-Tris HL5, harverst and add to tubes, and then place them between two foam 15ml conical tube racks to insulate, tape the two racks together and place the whole rig in the -80 and let slow freeze at least overnight before putting them in boxes. We freeze 0.5-1ml per tube and with various amounts of cells. I haven't seen any evidence you need high cell titer, although we usually have it as we have also frozen low titer cells this way. To revive, we quick thaw and place the contents of a tube into 10mL of HL5 in a dish, and as soon as they are attached of settled, change the media with fresh to get rid of the DMSO.
Dave
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Dr. David Knecht

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>For freezing DMSO's we set up the tubes in an ice bucket change the media in the plate to Bis-Tris HL5, harverst and add to tubes, and then place them between two foam 15ml conical tube racks to >insulate, tape the two racks together and place the whole rig in the -80 and let slow freeze at least overnight before putting them in boxes. We have found that a "Mr. Frosty" cell freezer gives enormously better viability than this method. Various outfits sell them - you fill them up with isopropanol and they standardize the rate of cooling.
Robert
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Robert Insall