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I'm volunteering for a biohacker lab - biocurious in Sunnyvale. The have a pretty good set of equipment - gel boxes, incubators, but they don't have a -80°C freezer yet.

I'd like to set up some glycerol stab stocks of some E. coli strains, but i'm told even untransformed (i.e. no plasmid) bacteria will get funny after a while at -20°C. It would also be good to hear how long you can keep a plasmid transformed strain at -20°C.

Can anyone be specific about what happens and how long it takes when you store them at -20°C for long periods of time?

I have done a little reading for competent cells and I guess they stay active for about 4 days without a -80°C freezer.

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  • $\begingroup$ so i'm getting that everyone thinks that the bacteria will simply die at -20C. I asked this question because I was wondering if it was apocryphal. Everyone 'knows' that -80C is the best temperature for glycerol stabs, but i'm wondering if its 100% necessary, esp over something like 3-4 months. Some good suggestions though. I will try it with some stabs and get some protease deficient strains as well. $\endgroup$
    – shigeta
    Commented Feb 28, 2012 at 2:44
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    $\begingroup$ You can also start keeping transformed cells (with some drug resistance marker) at -20°C, and plate some of them every day and see how quickly they will lose the plasmid. You can do the same with untransformed competent cells. $\endgroup$
    – Gergana Vandova
    Commented Feb 28, 2012 at 21:46
  • $\begingroup$ some great points here. If its okay i'm going test the answers to see who is right before picking one. if anyone is in the peninsula area of the sf bay area, you are welcome to come to biocurious and check it out. ;) $\endgroup$
    – shigeta
    Commented Feb 29, 2012 at 21:05
  • $\begingroup$ I might do that:) Can you email me ([email protected]) with some more details about it? $\endgroup$
    – Gergana Vandova
    Commented Feb 29, 2012 at 21:33
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    $\begingroup$ Mother nature's got you covered. -136 ˚F (-93.2 ˚C). Locate you samples: in a small valley between two peaks on the East Antarctic Plateau. ~Dec 10 2013; ;) Maybe not what you were looking for, but for this one point in time you could have just put your stabs out on the back porch. Now that is a cold day! blogs.scientificamerican.com/observations/2013/12/10/… $\endgroup$
    – mike
    Commented Dec 10, 2013 at 9:29

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I think that cells get damaged at -20°C not because they are stored for a long period of time, but because they undergo cycles of thawing and freezing (the ice crystals that form damage them).

I never keep cells at -20°C. I store them at -80°C in 50% glycerol. The idea behind the glycerol is that it serves as cryoprotecor. It forms hydrogen bonds with the water molecules and thus it disrupts the water-water hydrogen bonds. As a result, it disrupts the crystal formation of ice, and the cells don't freeze. The freezing temperature of glycerol is −37.8 °C (60-70% glycerol in water), which means that at -20°C glycerol does not freeze and thus it cannot protect the cells from damaging from ice crystals formation.

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    $\begingroup$ A good -20°C freezer should not cycle between freeze-thaw. This is a problem with frost-free freezers, warming up to near O°C. Indeed, the glycerol is the trick for preservation of membrane integrity. Decades ago, researchers stored their bacterial cultures at room temperature, in the dark, in what were called stabs. $\endgroup$
    – Larry_Parnell
    Commented Feb 27, 2012 at 20:39
  • $\begingroup$ When I said a freeze-thaw cycle, I meant when you take the frozen sample from the freezer, do something with it (it thaws), and then put it back in the freezer (it freezes). $\endgroup$
    – Gergana Vandova
    Commented Feb 27, 2012 at 20:42
  • $\begingroup$ This is an interesting answer - if it's only the ice crystals, then one could use 40% glycerol and the solution would freeze solid while still being mostly just as good a cryoprotectant. en.wikipedia.org/wiki/… I tend to think that there may be some more specific answers out there though... I'll try a 40% glycerol stock in LB (or whatever it takes to freeze solid and see how this works. $\endgroup$
    – shigeta
    Commented Feb 28, 2012 at 2:39
  • $\begingroup$ @shigeta I didn't know that proteases are active at -20°C (leonardo's answer). But it is worth the shot (at least short-term) if you don't store the cells too long. $\endgroup$
    – Gergana Vandova
    Commented Feb 28, 2012 at 2:44
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Gergana covered the "why" part of your question. +1

If all you have at the moment is a -20°C and mostly what you want to store is E. coli harboring plasmids, I'd recommend preparing plasmid DNA and storing that at -20°C. The DNA will stay stable in the medium term, and you can re-transform into E. coli once you've got your -80°C freezer up and running. And ideally you want both DNA stocks and bacterial stocks on hand - you never know when a freezer will go down.

For your untransformed strains, you could make agar stabs or plates to store at 4°C. I would re-streak them every 1-2 weeks, grow overnight at 37°C, and then put them back in the fridge.

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The answers already posted about glycerol being a cryoprotectant and ice crystals are correct. The other thing to consider is that enzymes still retain some activity at -20°C and though the reaction rate is slower, the cells will degrade until they are no longer viable. I always store glycerol stocks of bacteria at -80°C.

Many common E. Coli strains commercially used for cloning are not protease-free and these are the enzymes that are responsible for cellular degradation.

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    $\begingroup$ Which enzymes are active at -20degrees? $\endgroup$
    – Gergana Vandova
    Commented Feb 28, 2012 at 1:11
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    $\begingroup$ Proteases. Just edited my post. $\endgroup$
    – user560
    Commented Feb 28, 2012 at 1:26

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