freezing and storing bacteria

[Mega]

As I finally have my glowing bacteria, I would like to store some of them in the freezer in case that the Plasmid is thrown away by the bacteria.

How do you store your bacteria?


Take a toothpick of bacteria, put it into glycerol and then into the fridge??

Or just agar in an eppi, and the bacteria on agar. In the fridge-> ready?


Thanks,

[Jordan Miller]

Nicely done!

The standard LONG term storage is storage in 50% glycerol in water and placed at -80ºC. -20ºC may be fine (standard household freezer temperature) but household freezers fluctuate in temperature to go through a defrost cycle which is not good for your bacterial stocks.

In the fridge the bacteria will continue to grow but very slowly -- the upshot is that they will lose your plasmid within a month or two. Don't store them in the fridge.

good luck!

jordan

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[Derek]

I put my bacterial stocks into a foam shipping container with a coldpack in it, and put that into the freezer. Grow bacterial culture to mid-log phase (just so that the greatest number of cells are viable and actively dividing), then dilute 50/50 with glycerol, and toss it in the freezer. I usually try to renew stores every year, just to make sure they're OK, but I've restored stocks as old as 3 years without any issue.

--Derek

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[Avery louie]

It seems like you want to maintain the plasmid, not the strain!  Consider doing a plasmid extraction, and freezing/drying the plasmid.

--A

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[Avery louie]

Also, send us some pictures!  I would love to see your work.

--A

[Andreas Sturm]

Yeah, that too... (strain & plasmid)

I'll google how to do that and maybe I'll do a huge miniprep to get some (or a lot of ;) ) plasmids.

I don't know why the bacteria in the middle of the dish don't glow strongly so that you can see it. Maybe they have consumed all of the sugars of the agar?


That would be an idea: whenever I need glowing bacteria, I do a transformation... Well, if I can grow me enough plasmids, why not?
But therefore I'll need EDTA, SLS, etc. ...
Once or twice I surely can try at university, but someday I'll have to get my own chemicals :)

2012/5/9 Avery louie <inact...@gmail.com>

[Cory Tobin]

> I don't know why the bacteria in the middle of the dish don't glow strongly
> so that you can see it. Maybe they have consumed all of the sugars of the
> agar?

I would re-streak the bacteria on a new plate to get individual
colonies. If you're unfamiliar with quadrant streaking, check this
out http://www.umsl.edu/~microbes/streakplates.pdf It's basically a
way ensure that you get some individual colonies.

Also, if you will be transforming more bacteria with the same plasmid,
it's helpful to make a couple of serial dilutions and plate each
dilution out on different plates so you don't get the giant mass shown
in your image. If you take the transformation and dilute it 10x in
LB, then dilute that 10x in LB, then plate all three dilutions (1x,
10x, 100x), one of those plates will probably have individual
colonies. For example, here is a 100x dilution of a GFP
transformation in E coli plated on LB and illuminated with UV
http://i.imgur.com/mWPk1.jpg The image sucks (taken with my phone)
but you get the idea - single colonies.

-cory

[Jeswin]

On Wed, May 9, 2012 at 3:03 PM, Andreas Sturm <masters...@gmail.com> wrote:
> Yeah, that too... (strain & plasmid)
>

For the bacterial cell culture storage, I add approximately 300uL
glycerol to 1.5mL tube and add 500uL from overnight inoculation
(usually from our 100mL maxiprep culture). I vortex for complete
mixing and immediately rush to the -80C and freeze it down. I hear
glycerol will destroy the cells unless frozen down.

Plasmid: you have to extract the DNA somehow and elute in TE buffer.
Store in -20C freezer.

[medminus9]

In the fridge the bacteria will continue to grow but very slowly -- the upshot is that they will lose your plasmid within a month or two. Don't store them in the fridge.

 In case you have a non frost-free fridge (the one's that have ice deposition), the freezer won't defrost regularly and can thus be used for storage. You bacteria should easily withstand upto 5 freeze-thaw cycles.

Good luck!

Harsh Sethia