Will E. coli Grow Over a Weekend on a AMP/LB Plate?

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In summary, Ian and the other participants in this conversation discuss the consequences of leaving E. coli on a AMP/LB plate over the weekend. They mention that the bacteria may survive if the plate is inverted and incubated for no more than 96 hours. They also discuss the appearance of satellite colonies and the effects of AMP on bacterial growth. The conversation also touches on the use of E. coli for cloning and the process of isolating a vector with a resistance gene.
  • #1
Monique
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Ian? What happens if you leave E. coli's out to grow on a AMP/LB plate overweekend? :frown: I left my plate in the stove and they are REALLY important clones and I don't have the key to the lab! :eek: They'll live right?
 
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  • #2
It should live only if your plates were inverted and your incubation does not go beyond 96 hours. If your plates were not inverted, the plates migth dry out and it migth kill your bacteria. Be sure you are in early morning and restreak your colonies on fresh LB. Do you have frozen permanent or any sort of backup (e.g. plate in the fridge)?
 
  • #3
96 hours huh? *relief* this wás actually my backup, I plated 13 different most promising colonies on this one plate so that I could plate them out once the result of the colony pcr on 66 colonies came back.. out of the 66, three finally came out with the right thing.. which were on that plate :rolleyes: (and yes, I did a blue/white screening) it's a really difficult construct so I wouldn't want to do the whole thing from start again :smile:

What are exactly those satellite colonies that come up if you grow them too long? I know the amp gets broken down by the resistant colony, but do those satellite colonies just come dropping out of the air?
 
  • #4
Amp inhibits the growth of bacteria but does not kill them. By the time amp is broken down some bacteria have survive the starvation and start growing. This is why satellite colonies are smaller. The satellite colonies will only appear during your first round of selection. Not satellite is present after you select and streak for isolation your desired clones.
 
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  • #5
oh really, I didn't know that..
thanks Ian :biggrin: :smile:
 
  • #6
Monique, have you considered calling somebody who has the lab key to let you in, or, alternatively, who can take your plates out for you if they are in the lab this weekend? On most campuses, you can call security to let you in if you usually are supposed to have access and have misplaced your key (or locked it in inside the lab, as I've done to myself several times). Just calling the lab and leaving a message usually does the trick...I've never been in a lab where SOMEBODY wasn't working over the weekend ;-) I know that doesn't directly answer your question, but it may solve the problem nonetheless. Or email your PI. Everyone has done something "forgetful" in the lab at some time in their life, and this is a pretty minor one, so I'm sure someone will take mercy on you and let you into retrieve your plate. Good luck with those colonies.
 
  • #7
What are you guys talking about ?
 
  • #8
I think what's going on is that Monique left some cultured E.coli (a bacterial) in a stove, and can't access them for the weekend, and was wondering if they would survive. Apparently they will as long as the plate is inverted, so the colony on the plate stays moist.

AMP here stands for ampicillin (an anti-biotic) and not adeno-mono-phosphate (a metabolite). LB is an agar plate, in which E.coli is grown on.Something like that anyway :p

Anyway, a question on my part - Can AMP kill bacteria or will it only stop growth? Or is it only able to kill cells that are actively dividing?
 
  • #9
:smile: I called but there's no one there :rolleyes:
I could go there, but that'd cost me 3 hours total traveling time.. and I'd have to make a fool of myself searching through the whole academic hospital for a guard with a key.. just for a silly plate :redface:

As Ian said, I'll restreak them on monday :)
 
  • #10
Jikx said:
Anyway, a question on my part - Can AMP kill bacteria or will it only stop growth? Or is it only able to kill cells that are actively dividing?
Good question, what is the molecular basis on which ampiciline works? From Ian's statement I understand it is biostatic and not biocidal.
 
  • #11
The beta-lactam group of antibiotics is only bactericidal on growing cells. These antibitotics inhibits the formation of peptidoglycan cell wall. Once the cell wall is destroy the bacteria lose their shape and cannot regulated the desired osmotic pressure. The cell lyses due to acumulation of water in their cytoplasm. In vitro, you can stop the lysis of gram positive by using special media and you end up with bacteria lacking a cell wall and the bacteria are all round. Cells that are not actively growing will not be killed.
 
  • #12
Monique, what are you doing with the E.coli culture? Are you growing some that are resitant to amp?
 
  • #13
No, it is a non-pathogenic strain of E. coli which normally is not able to grow in the presence of ampiciline. What I made is a certain contruct in a viral vector, a piece of dna, which the bacteria have taken up. In this contruct there is a piece of DNA that contains the restistance gene against amp. So only the bacteria with the construct will be able to grow. I can later isolate this vector with the insert, a plasmid, from these bacteria.

So the construct that is made can either contain a gene or portions of DNA that regulate genes. The bacteria just serve to amplify it, so that it can be isolated and put back into another celltype to evaluate its effect.
 
  • #14
Monique said:
No, it is a non-pathogenic strain of E. coli which normally is not able to grow in the presence of ampiciline. What I made is a certain contruct in a viral vector, a piece of dna, which the bacteria have taken up. In this contruct there is a piece of DNA that contains the restistance gene against amp. So only the bacteria with the construct will be able to grow. I can later isolate this vector with the insert, a plasmid, from these bacteria.

So the construct that is made can either contain a gene or portions of DNA that regulate genes. The bacteria just serve to amplify it, so that it can be isolated and put back into another celltype to evaluate its effect.

In other words you are doing cloning with probably DH5 alpha with a pUC derivative :wink:
 
  • #15
iansmith said:
In other words you are doing cloning with probably DH5 alpha with a pUC derivative :wink:
Close, cloning with DH5 alpha and pGEM(-T) (among others: pGL3, pcDNA3.1) :)
 

1) Will E. coli grow on an AMP/LB plate over a weekend?

Yes, it is possible for E. coli to grow on an AMP/LB plate over a weekend. The growth rate of E. coli can vary, but given optimal conditions, it can double in population every 20 minutes.

2) What is an AMP/LB plate?

An AMP/LB plate is a type of growth medium specifically designed for E. coli bacteria. It contains two main components: LB (Lysogeny Broth) and AMP (Ampicillin). LB provides essential nutrients for the bacteria to grow, while AMP is an antibiotic that only allows for the growth of bacteria containing a specific gene that makes them resistant to the antibiotic.

3) How does AMP prevent the growth of non-resistant bacteria?

AMP works by targeting the cell walls of bacteria and inhibiting their ability to reproduce. Bacteria that contain the specific gene for AMP resistance are able to produce an enzyme that breaks down the antibiotic, allowing them to continue growing and reproducing.

4) How long does it take for E. coli to grow on an AMP/LB plate?

The time it takes for E. coli to grow on an AMP/LB plate can vary, but under ideal conditions, it can take anywhere from 12-24 hours. Factors such as temperature, humidity, and the amount of bacteria initially added to the plate can also affect the growth rate.

5) Can E. coli grow on an AMP/LB plate if it is left out at room temperature?

Yes, E. coli can still grow on an AMP/LB plate if it is left out at room temperature. However, it may grow at a slower rate compared to being incubated at a warmer temperature, and there is also a higher risk of contamination from other bacteria or fungi present in the environment.

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