Long Term Bacterial Storage in -20C Freezer...glycerin stock?

[Sebastian Cocioba]

Has anyone had success storing bacteria for the long haul (1yr+) in standard freezers (-20C)? I know one makes a glycerol stock for -80C deep freezers but would the same be possible in -20C? Since it is warmer, what would the theoretical media refresh intervals look like? Monthly? Quarterly? Growth rate? Inhibitor additions? A bio-hacker might not have access to such a deep freezer but we all have freezers in our fridges. Thanks again and as always, happy biohacking!

[Mega]

I would estimate not taking too much glycerol. On the one hand, as it is not so cold, it will freeze slower. So less damage by cristallization. And glycerol is poisonous for the cells, I read. 

I kept bacteria on petri dishes (lb amp) for months in an old fridge (4-8°C). Then recultivated them without any problems. Actually, 99% of the bacteria can die and it doesn't matter. One must survive and within days you have 100000000s of them.

[Matt Lawes]

Another storage option is an agar stab. That lets you store at room temp indefinitely if you para film the cap of the vial properly. I can dig up a recipe / method if requested. >matt

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[Sebastian S. Cocioba]

That would be great! A room temp bacterial library would be ideal. Thanks.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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[shamrock]

A better strategy would be agar stabs, I've stored cultures (E. coli and S. cerevisiae) as stabs for six months without subculturing and my guess is you could go longer. The trick is slowing metabolism way way down by keeping the cells under anaerobic conditions and in the cold. To make a stab prepare sterile growth media (LB is what I typically use for E. coli) with agar and pipette about 1 ml into a 2 ml screw cap tube. I place the tube on an angle when I prepare the tubes so that the agar has a slanting surface when it hardens. When you inoculate stab the culture down into the agar and then let it grow for a day or so at 37 until you see visible growth. Tighten the cap and seal it with para-film if you want then store it at 4 degrees.  To revive just stab down into the culture with a sterile loop and away you go.

[Sebastian S. Cocioba]

Thanks so much! You should post this on open wetware protocols. :)

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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[Matt Lawes]

Nice protocol from Shamrock for agar stabs. Here's a bit more of a recipe:

Makes enough for 100 stabs.

0.6 gm Agar
1 gm nutrient broth powder
0.8 gm sodium chloride
100 ml deionized water

Autoclave in a 150 - 200 ml bottle. Dispense 1ml into 2 ml screw cap vials. The stab agar stock bottle can be microwaved later to melt if you don't need / want to aliquot all of it at once.

Note: NB and LB are similar ..... straight substitute ... some LB mixes already include salt, in that case adjust recipe above. Salt level should be adjusted in general to the needs of your bacterium. Above recipe is standard E. coli / Salmonella mix.

>matt

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[Cathal Garvey]

I gather they are still best kept in the fridge after being allowed to
"take" at room temperature. Also, apparently survival is improved,
whether freezing or just chilling, by cooling the cells to room
temperature for a while before freezing (bacteria, not mammals, that
is): this allows expression of protective "cold shock" proteins.

Again, sorry if I can't provide the citation for that one. I can't
recall in what context I stumbled across it, but it mostly applied to
-80 storage. Probably equally true of -20 storage.

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[MikeyJ]

ATCC seem to think that it's a couple of months max at -20 (but that must depend on the bug) and have some manuals http://atcc.custhelp.com/app/answers/detail/a_id/976/~/long-term-storage-of-cultures . As an alternative to glycerol, which I've had toxicity problems with in the past, is 10 % (w/v) skimmed milk powder.  It used to work really nicely, is easy to get hold of and the powder lasts for ever.  Also, I hate pipetting glycerol, milk is easier.  

I would watch out for frost-free fridges or freezers if you're using a home one, the temperature cycles and will probably mean you have to subculture more regularly to maintain viability.