10 replies to this topic

[Trof]

I didn't do a serious work with bacteria for years so I'm going to ask some very basic questions, sorry.

How long will E coli (Stbl2 strain) survive in fridge?
I tried to google it, but all hits were about people asking how to kill them, I want to keep them

I transformed them with my plasmid and keep them with closed caps on 4 degs from yesterday. I need to validate my plasmid first by sequencing the complete insert, which will take several days. So will they be good for say 4-5 days?

Other option is to make a glycerol stock, but I stored them in fridge from yesterday so I would probably need to grow them fresh and there are 20 of them. I'd like to make a glycerol stock from the verified one(s), after validation and making a midiprep.

Also I couldn't find our protocol for glycerol stock, so I searched and is it I just take 150ul of glycerol (15%) and 850ul of bacteria, mix and snap freeze in dry ice and store at -80? And then to regrow, just thaw them on ice and put into liquid media (midiprep)? Or spread on a plate first?

Thanks in advance.

[hobglobin]

Bioforum's protocols has some good protocols for it, if I remember right I used similar protocols, and especially a higher glycerol concentration (60% and more).
And E. coli in fridge for a few days should work, though I would do it as short as possible, because of possible contamination problems especially if the antibiotics are gone and more important, the bacteria might get rid of the plasmids after some time.

[phage434]

Liquid cultures die much more quickly than colonies on plates.  I'd recommend plates (or stabs) for even temporary storage.   Avoid ampicillin as an antibiotic, since it degrades quickly an is bacteriostatic.

[Ameya P]

Trof,

Whenever you need to go back to your glycerol stock, you can take it out (not even thaw it completely) use your loop and streak them out on a plate. You can then use the plate for making liquid cultures.

If I am not wrong, & hob and phage will correct me if I am, bacteria taken off a glycerol stock do not survive, if you put them in liquid media. Some sort of " media shock" theory. I don't quite remember now.

[phage434]

I inoculate liquid cultures from glycerol stocks.  It's not that the cells die, but that streaking out for single colonies on a plate is good practice.  A mixed culture in a glycerol (intentional or unintentional or contaminated) can give you unreliable results in an experiment.  A single colony can at least be analyzed for correct morphology, and with the correct plate(s) for a phenotype.

[Trof]

Thank you all. I stored the original plates in fridge as well, but verified only the selected minipreps, so after verification I need to use the rest of miniprep. Sadly I can't help but use ampicilin, the plasmids we got from other lab have Amp^r only and that 10x more expensive ampiciline analogue I heard of is out of question.

[lyok]

phage434, on 02 September 2011 - 04:03 PM, said:

Liquid cultures die much more quickly than colonies on plates.  I'd recommend plates (or stabs) for even temporary storage.   Avoid ampicillin as an antibiotic, since it degrades quickly an is bacteriostatic.

How come liquid cultures die more quickly?

And what happens then when ampicillin breaks down? What does it form then that it becomes bacteriostatic for the bacteria you want?

[phage434]

Just an observation, I don't know the specific answer.  Presumably cells grow more quickly and exhaust the medium more rapidly.  Ampicillin is bacteriostatic, so when it is degraded by beta lactamase, secreted by resistant bacteria, it goes away and allows non-resistant cells to continue growing.

[lyok]

phage434, on 02 September 2011 - 04:03 PM, said:

Liquid cultures die much more quickly than colonies on plates.  I'd recommend plates (or stabs) for even temporary storage.   Avoid ampicillin as an antibiotic, since it degrades quickly an is bacteriostatic.

phage434, on 03 September 2011 - 09:32 AM, said:

Just an observation, I don't know the specific answer. Presumably cells grow more quickly and exhaust the medium more rapidly. Ampicillin is bacteriostatic, so when it is degraded by beta lactamase, secreted by resistant bacteria, it goes away and allows non-resistant cells to continue growing.

Ah ok, but reading your first post it looked like you wanted to say that the break down products of ampicillin itself became toxic.
Its just because the ampicillin is broken down that it has no effect anymore on other (unwanted bacteria)?

But then I have 2 questions:

- normally there shouldnt be any other bacteria anymore in the samples right? SInce they would have been dead because you started with ampicillin. So if you do get other bacteria to grow, it means you contaminated it after the start, right?

And second question
- what with other antibiotics? Arent those not broken down then ? Just like the ampicillin?
(meaning they would also be useless after a while?)

[phage434]

Unless you were well trained in a tissue culture lab, there will be bacterial contamination in your samples.  The only question is how much.  Also, bacterial cells lose plasmids, so a cell can start out resistant and become non-resistant.  These cells typically grow faster than resistant cells.

Other antibiotics are less prone to this, although other degradation pathways (both biological and chemical) are at work.  They are slower.  Carbenicillin can replace ampicillin as a selective antibiotic, and is substantially more stable.

[lyok]

O k, I see what you mean.  
But even if you have a lot of contamination, the ampicillin would kill off all the unwanted bacteria at the start right? So if I would not open the vials again, the only unwanted bacteria after a while would be the ones that had the plasmid first and then lost it? right?

Or is it possible that other bacteria stayed in the vial and survived? maybe sporeforming bacteria? Or is this a long shot?