Store your bacteria and plasmids dry at RT with the magic of trehalose! No -80 needed!

Published: April 9th, 2020   Last Modified: November 5th, 2020

For many people, their -80C freezer is one of the most critical pieces of lab equipment. It can be your life’s work, all packed up in 3x3ft footprint. Your glycerol stocks, competent cells, tissue samples etc. Getting a call that your -80 is in a rapidly expanding puddle of water is no fun, to put it lightly.

We put up with it, however, because what other choice to we have? As it turns out, quite a bit! There are a few ways in which you can lessen your dependence on the -80, and one that I’ve been investigating is preserving E.coli and plasmid DNA dry at room temperature! Let’s see how you can add this trick to your sample preservation toolkit!

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Trehalose is a non-reducing disaccharide which is widely used both inside and outside the lab. In the biological world, its a carbon source and a cryo/osmo protectant, and is used in the health, food and medical industry. I won’t go too deep into the background of trehalose because it’s already been done better in a review by Nishant Kumar Jain and Ipsita Roy in 2009.

Basically, it seems like trehalose is a miraculous molecule when it comes to stabilizing biologics. I was clued in a few years ago when I was skimming patents, and the use of trehalose to stabilize basically everything kept popping up. I filed the ideas away as something for future Alex to try. I realized when I unearthed my time trail of plasmid stability that I have a need to dry things and preserve them, like a little squirrel hiding nuts for winter.

So, is it possible to store the organisms we work with dry? The following patent (https://patents.google.com/patent/US20030044965) describes the storage of various bacteria, including our friend E.coli, with trehalose. Basically, here is their 2X recipe:

2X Preservation Media
200 mM Trehalose
10mM CaCl2
10mM MgCl2
10mM ZnCl2

Their method boiled down?
1) Combine 1:1 with a overnight E.coli culture or other bacteria
2) Dry down in some manner (They legitimately suggest drying the bacteria just open to the air, how’s that for low tech?)
3) Store dry at RT somewhere
4) Reconstitute when needed. Media or water added to dry cells, plated or grown up in liquid media

Viability of cells drops by an order of magnitude upon drying, but after drying the cells are extremely stable, with negligible degredation after 5 months!!!

It…can’t be that easy, can it?

So, my setup? The cultures I preserved were E.coli carrying the pfu-sso7d plasmid, grown in LB Kan media overnight to full saturation. With this method you want as many cells as possible, early stationary is likely ideal. I combined the cultures 1:1 with 2X Preservation Media, and made 40 uL aliqouts in 1.5 mL tubes. I don’t have the patience to let the bacteria dry slowly in the open air…a Centrivap concentrator dries them in 30ish minutes at 37C. What do the little dried up bits of bacteria look like?

Hmm, after reading the trehalose review, I would have to agree, the pellet looks glassy. All your E.coli are trapped in a glassy, hard matrix and preserved. Give it a poke with a pipette tip and it crumbles to dust, there is very little moisture left there.

So, I stuck em in a box, stuck said box in a drawer, and I waited. I’ve been taking out dried samples and playing around with reconstituting them. Basically, you have two options:

1) Re-suspend the E.coli in 1mL LB, invert or shake for a few minutes, spin down and plate all the cells.
2) Re-suspend the E.coli in 1mL LB, add to media with antibiotic, grow overnight, use cultures like normal.

Okay, shut up and give the data already, I hear you, I hear you. Control, day 0, no drying, just added 1mL LB to 40 uL of the 1:1 mix and plates 20 uL of that 1mL.

Nothing to see here really, the take home is that the bacteria are happy and aren’t too bothered being mixed with trehalose and metal ions. What about the next day? Re-suspended as before, plated 20 uL.

Not too much viability lost, seems unusually high, but it’s possible at this stage the trehalose/bacteria syrup hasn’t reached its full glass state yet. What about 1 week? Did a couple of different tests here for fun, what if you let the bacteria recover in LB for more than a few minutes? Left is 1 hour in LB before plating 20 uL, middle is 3 hours, right is overnight.

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Very interesting, looks like with this method you want to either plate shortly after re-suspension OR let it grow overnight and then do what you need to do. Notice the sharp drop in viability when grown for only 3 hours. Cool, 2 weeks? Left is cells which were resuspended, inverted for 5 minutes, plated 20 uL. Middle are cells left to recover for 1 hour and 20 uL plated. Right is inverted for 5 minutes, centrifuged, plated all cells.

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Again, we see that it’s better to plate your cells right away than to try and recover them. This is likely that after desiccation, the cells are primed for a desiccated environment, not a liquid one. From this point on I switched to a more standardized protocol of resuspending in 1 mL LB, shaking at 250 RPM for 5 minutes at 37C, centrifuging to pellet, and plating the whole pellet. It seems that after rehydration, the bacteria tend to “sink” more than a normal liquid culture would, so plating only 20 uL is pretty variable if you only take from the top. 6 WEEKS?!?!?! Left is 20 uL plated after 5 minutes of shaking, right is all the cells after 5 minutes.

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See that variability with plating 20 uL? This time it gave a bunch of colonies. Plating all your cells is the way to go. That’s all the plate pictures I have now, a watched time course never…boils, so I have to let some time pass by. Take homes?

1) E.coli can be stored for long periods of time dry with 2X Preservation Media. Patent states at least 5 months, I’m at 6 weeks at counting.
2) Either re-suspend your cells and plate right away, or grow them up in liquid media overnight.
3) Re-suspend your cells, centrifuge, plate the whole pellet. Some variability when taking a small fraction.
4) If you’re plating right away, probably better to re-suspend in 100uL of LB or water

So…am I done with the preserving properties of trehalose? Tre-hell-no-s! *crickets*

I was also curious whether trehalose increased the shelf life and stability of plasmid DNA. As you recall, plasmid DNA in a plastic pouch does better than on filter paper. Is plasmid DNA in a pouch MORE BETTER with trehalose? I was able to keep my hands off these pouches for 3 months. The plasmid is DpnI, dried down in 20% trehalose, 5 mM Tris pH 8, 0.1 mM EDTA. I deposited 500ng per pouch. 50 uL of TE was added to each pouch, left to sit for 5 min, transformed 100 uL of DH5a with 1 uL of reconstituted plasmid. One sample I left at RT in my drawer, one went in the 37C incubator for accelerated aging, the last one was taped to the window facing the sun. So, RT (Left) vs RT + Trehalose (Right)?

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Yep, obvious increase in plasmid viability with trehalose. Remember, I plated 1 uL of 50 uL of resuspended plasmid…there’s more viable plasmid than you know what to do with. How about that accelerated aging, 37C for 3 months? 37C (Left) 37C+Trehalose (Right).

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Yup, increasing the temperature certainly speeds up the degredation of the plasmid DNA. Trehalose certainly protects the plasmid DNA significantly, pretty impressive, no? What about Sun (Left), vs Sun + trehalose (Right)?

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I was expecting a more drastic decrease in viability…maybe less sun since it’s been winter? Variability due to plating? Either way, trehalose helps out!

Anyway, enough trehalose for now, but I’m not done with it yet. I’d like to investigate making dry enzyme mixes, as described in the following paper by Bhadra et al. 2018. Soon, my friends, soon.

Also, competent cells can be stored dry with trehalose…very interesting.

If you’ve never seen or used a centrivap, here’s what one looks like:

Basically it’s a centrifuge inside a vacuum chamber with a heater element inside. If you can afford one, or can acquire one, GET IT. Easily one of the most useful but underappreciated bits of lab equipment ever. Often sold on ebay used.

7 MONTH UPDATE: Tested the viability of the cells after 7 months, unfortunately I had no growth on plates, either when I plated cells right away or growing them in liquid culture overnight. So, viable up to 6 weeks in my hands, then at some point during storage the cells degraded. I stored the cells in 1.5 mL tubes at room temperature, in the dark, in a cardboard box. The tubes were not parafilmed and there was no dessicant. More investigation is needed for reliable, long-term storage. I will be revisiting this story, for sure!