E.Coli growth at 4 degrees? - (Sep/15/2010 )

Hi,

I recently did a transformation experiment with some top10 E.Coli cells from Invitrogen, the transformation didn't appear to work too well but that is a different story.

I counted the number of colonies on the Ampacillin plates and put little black dots opposite each colony as I counted, I then wrapped the plates in Parafilm and placed them in the fridge. A week later I got some of the plates out again and saw many more colonies than I counted all over the plate and more than just the ones opposite the black dots, and these weren't tiny microcolonies, these were around the same size as the origional colonies.

Can E.Coli grow colonies at 4 degrees C? and if so, how then do I store plates for extended periods of time?

Ampicillin tends to break down more easily than other antibiotics. If you want to store your cells, I'd recommend toothpicking them onto a new plate, growing them up overnight, and then storing them at 4degC. Even so, you don't want to store them more than a couple of weeks that way without re-toothpicking.

philman on Wed Sep 15 12:31:16 2010 said:

On a very funny note phil, you have too many colonies on your plate......
We had a 4oC room at our univ. and I have kept(forgotten) plates for a month with almost no growth on them. Was the fridge working fine, while you were away??? or do you have a new student in your lab who might have put them out and put them back in the next day....?
I know there is no technical help from me, but I can't think of anything for both the problems you have mentioned here....

gt_ameya on Wed Sep 15 12:55:58 2010 said:

Yes I know I have too many colonies, I rather overestimated how many transformants I would get! I am re-plating today at a higher dilution! And The fridge should have been working fine, they were only left for a week. I might stick a thermometer in there in a bit to see how cold it actually is, it is a rather old fridge...

and I AM the new student, and I don't rememeber leaving them out

and yes I know Ampicillin is a bit crappy, I left my negative control plate in the incubator for 5 days once out of curiosity and it came out with colonies on it (although there were none on the day after transformation so the controls were still viable)

went ant tuck a thermometer in a flask of liquid that had been stored in there for quite a while, the fridge was only at 13 degrees!

I have turned this down now and hopefully the temperature will go to nearer 4 degrees by tomorrow, but an annoyng way to find out that it has been that warm for goodness knows how long

philman on Wed Sep 15 15:13:43 2010 said:

Thats exactly why u need fresh students every summer...... Unusual things happen to their experiments and you find out what is ACTUALLY going on in your lab.

gt_ameya on Fri Sep 17 04:47:21 2010 said:

I turned the fridge down to minimum and the temperature only went down to 9 degrees the next day... have grown suspicious of all the fridges in the lab now and have put a 15ml tube of tapwater in each one yesterday to check the temperatures later today... could be a problem...

This doesn't have anything to do with your refrigerator -- you're seeing satellite colonies arising on the plate.

When you plate a transformation mixture, it contains many cells that haven't been transformed, and (hopefully) some that have. Initially, these cells without a plasmid don't grow because of the ampicillin in the media. However, as the transformed cells grow, they secrete beta-lactamase (the enzyme that destroys ampicillin and confers resistance on the transformants). As this enzyme accumulates in the media and diffuses outward, the concentration of ampicillin drops, because the enzyme is destroying the ampicillin. Now the untransformed cells can grow, as the concentration of ampicillin remaining is too low to stop their growth.

So, yes -- E. coli does grow at 4 degrees, and you shouldn't store your transformation plates directly, rather you should pick your transformants to a fresh plate and store that.

philman on Wed Sep 15 12:31:16 2010 said:

Typically, these colonies only appear when you select on ampicilline, as this gradually degraded in the fridge.
I think the best way is simply restreak your colonies on a new plate with ampicilline and store those plates. After all, colonies will never appear if you haven't plated them there!


Good luck!