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bacteria grow on plate but not in broth


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#1 bsg2004

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Posted 12 October 2004 - 09:56 AM

Hi, there, I am confused by what happened to my bacteria. We got several tubes of stuffs from another lab. As we had no idea whether the tubes contained plasmid or plasmid transformed cells, we streaked the stuffs in the tubes onto Agar plate with Amp. A lot of colonies appeared on the plates one night later. However, the colony picked from these plates did not grow in LB or NZY broth with Amp but did grow in Amp-free broth.

Anybody can tell us what is going on here?

Thank you very much.

#2 vetticus3

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Posted 12 October 2004 - 10:49 PM

is the concentration of amp the same as on the plate, as in the broth?

#3 Squawks

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Posted 13 October 2004 - 02:23 PM

Hi, bsg2004 - how old are your agar/amp plates? Ampicillin has a reputation of wearing off as time passes; it's a good idea to use freshly prepared plates or recently poured ones. I myself use carbenicillin over amp.

Try doing a miniprep on the cultures that grew from the amp-free broth and see if you can retrieve any plasmid...if not, your colonies are most likely contaminants. Some strains nevertheless still carry plasmids naturally, however, so don't put all your faith in this method's results.

#4 bsg2004

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Posted 13 October 2004 - 02:48 PM

Thank you all. I appreciate it.

The broth and the plates were freshly prepared, and the Amp concentration is the same.

I picked colonies from the Amp-LB plates and grew them in Amp-free LB broth. As expected, the bacteria grew very well.

I will do a miniprep to see what's going on. Maybe, the colonies were nothing but just contaminants.

More suggestions are welcome.

Thank you.

#5 refadl

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Posted 13 October 2004 - 09:45 PM

Hi ... are you suren that the ampicilin powder still is okay ?
can you get some amp powder from another lab ? ?
Had the same problem picking a clone and growing it in LB ... it did grow on the LB agar plates (where as we found out afterwards the amp was not working anymore) but it did not grow in the LB with freshly added Amp.
So maybe your amp powder just has expired ... best regards
refadl

#6 bsg2004

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Posted 14 October 2004 - 03:53 PM

Hello, after a few tried, now it seems apparent that there was something wrong the Amp when the plates were prepared.

Thank you very much.

#7 leahf

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Posted 17 October 2004 - 04:24 AM

Hi
this is a long shot but...
the ampicillin could be less efficient in a plate than in broth. for example, say your cells are "partly resistant", that is, expressing a very low concentration of the anti-ampicillin protein. in a plate it might not matter very much, if the colony divides faster than the rate of ampicillin diffusion through the agar - that is, even a low degradation rate might be enough to allow the colony to form. in the broth the ampicillin concentration is homogenous and a low degradation rate may not be enough.
to test this - maybe decrease the ampicillin concentration of your broth?
another long shot:
it could be that only some of your colonies are truly resistant and the rest are satellite colonies (enjoying the benefit of having the amp degraded by the resistant ones) and you have had the bad luck to pick the satellite ones. in that case there should be a size difference between them, with the resistant ones bigger than the others. or, you could try transferring the entire plate into broth+amp (pour in a couple ml of broth, rub gently with drigalski and transfer to growth vessel) and see if anything grows, and then replate it.

good luck!

#8 bsg2004

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Posted 29 October 2004 - 12:36 PM

After combined the suggestions from several friends above, I did the following things: pick a streak of colonies (instead of a single one) from the plate and grew these colonies in amp free or lower amp LB broth via mini-prep, then streak the cell cultures on plate prepared with amp concentration ranging from 25-100 ug/ml. Repeat one to three times. Finally, I got the single colony that was able to grow in high amp LB broth! The plasmid isolated is in the right size.




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