Best storage methods for strains in -20 freezer?

[Patrik D'haeseleer]

Hi all,

The gold standard for long-term storage of strains is glycerol stocks in a -80 freezer. Or even an ultra-low at -150C, or liquid nitrogen at -196C.

However given that most DIYbio labs don't have access to those kinds of resources - what are the best recommendations for storage in a -20C freezer?

50% glycerol? 15% glycerol? 10% skim milk? Stab cultures?

Opinions / best practices wanted!

Patrik

[Koeng]

I've actually experimented on this :D

https://groups.google.com/forum/#!searchin/diybio/4$20months/diybio/u4mxuMNTyMw/VbgwOQA5IUwJ

That was a couple of months ago, so I'll check it again if you want me to see. Essentially, I tried it in -20 at 10%, 20% and 40% glycerol and tried to recover cells from that. They all worked. It seems like storing cells is pretty robust even in these conditions (for 4 months, so far)

I usually just put in 80% glycerol at half the freezing stock (250µl of 80% glycerol for 500µl of cells) and that's worked. To my knowledge, I'm the only one who has done a controlled experiment on this. It'd be great if someone else could put up their research or start their own, I would like this to be reproducible so I can recommend it! I'm working on a standard part distribution for DIYbiologists and it'd be great to have -20 storage protcols

(It also should be noted that I used E coli. S cerevisiae may be different because yeast freeze differently and are more prone to lysis (they just die more in stocks) and B subtilis doesn't even need freezing storage. I store B subtilis on paper) 

-Koeng

[Patrik D'haeseleer]

Excellent! Yeah, it would be great if you could keep checking those every once in a while. I have high hopes fro using skim milk as a cryoprotectant as well. - that apparently worked even better than glycerol in reviving cultures that had been defrosted during hurricane Katrina:

Skim milk enhances the preservation of thawed -80 degrees C bacterial stocks.

I would also like to start a little culture collection at Counter Culture Labs. We already have the pigmented bacteria set from Carolina (Micrococcus luteus, Serratia marcescens D1, Sarcina aurantiaca, Rhodococcus rhodochrous), and we want to start experimenting with a bunch of cheese making cultures for the Real Vegan Cheese project. So we'll have plenty of non-E. coli to test storage methods on.

Patrik

[Koeng]

Alright, I'll recover them tomorrow!

I don't think I have any dried skim milk at lab, otherwise I would try it. Glycerol is very easy, but I'd be very happy to see some protocols on skim milk to possibly use in the future! Glycerol seems very easy to use, but then again I don't have any experience using skim milk

I think it would be interesting if biohacker spaces got together and each bought a -20 freezer for redundant storage of cultures from other biohacker labs. Not only would it allow for everyone to keep their cultures safe from a freezer malfunction, but the other labs also get free strains to use in their research. Win-win all around

-Koeng

[Patrik D'haeseleer]

On Monday, February 9, 2015 at 10:40:56 PM UTC-8, Koeng wrote:

I think it would be interesting if biohacker spaces got together and each bought a -20 freezer for redundant storage of cultures from other biohacker labs. Not only would it allow for everyone to keep their cultures safe from a freezer malfunction, but the other labs also get free strains to use in their research. Win-win all around

Agreed. Counter Culture Labs has a -40 freezer, which could be a great resource for something like this. We also have a centrifugal evaporator system available that we could use to lyophilize strains for room temperature storage and transport. Maybe we should apply for a grant from ASM to set up a little DIYbio culture collection...

Patrik

[Koeng]

I recovered the cultures. All worked.

To recap, I had cultures with 40%, 20% and 10% glycerol. All were created on 6-25-14 and placed at -20 in a frequently used box. 

When I took the cultures out, 40% was completely aqueous. 20% was partially frozen, and 10% was barely frozen (similar to a very fine snow cone). I vortexed them all for a few seconds, which mixed them up a bit. Still, after that there was cells stuck to the bottom. The 40% I was able to mix up more, and after pipetting a few times in and out I got 20µl near the bottom (higher concentration of cells) into a culture tube. I did that with the others, although less efficiently of course because they were frozen. 

10% looked to have the highest cell growth rate after, but that could just be an error on my hand on perhaps taking cells too close to the bottom. 20% and 40% were closely matched in their visible RFP expression. All had nice cell growth.

My recommendation then would be 40% for now: the cells have stayed good and it being aqueous allows for you to get cells out quicker and easier. Yes, this is not a very well controlled experiment, but for simply storing cells it might be enough

I'll email you on the DIYbio culture collection! Perhaps we could get something worked out.

-Koeng

[Nathan McCorkle]

On Thu, Feb 12, 2015 at 1:19 PM, Koeng <koen...@gmail.com> wrote:
> I recovered the cultures. All worked.

Great work!

[Koeng]

UPDATE:

After another day, 10% has a much greater amount of RFP than both 20% and 40%. At the current time, I don't have an explanation, but I could try a few experiments next week if anyone is interested

[Mega [Andreas Stuermer]]

Maybe if the cells are frozen, proteases cannot work. If you add too much glycerol they domt freeze and emzymes may retain residual activity?

[Mega [Andreas Stuermer]]

I assume you plated on antibiotic free agar? Then maybe DNAses?

[Koeng]

Nope. I added cells directly to a new liquid culture with ampicillin. I'm not sure how good my amp is though, it's been in the fridge for 4 months now, I need to make a new stock