E.coli culture contamination - (Sep/08/2006 )

Hi all!
These days I have lots of problems with my bacterial cultures for protein expression. My problem appears when scaling up the culture (from an O/N) to 1 liter; The cultures start to grow normally but when they reach certain O/D (0.6-0.8) just at the moment of induction with IPTG th cell density of th culture starts to decrease and foam appears- the cells begin to lysate;
I am not sure of why does it happens (and why my o/n grow without problem)
Could anybody help
Thanx a lot!

rumo

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You don't want to hear this, but it sounds like phage contamination. The medium used for the o/n and the bulk culture may be different. The flasks are certainly different.

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That's the same coclusion as mine, phages; but some points made me doubt, like the fact that the O/N grow... is it really possible that the contamination is localized in the big flasks? ( they are washed and autoclaved periodically).
So then, which is the better way to get rid of them? work in a UV chamber?
Thanks!

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If you think you have a phage you can eliminate it by checking that all of your materials are autoclaved prior to use. If you are having trouble growing in a shake flask, then either you are adding the phage when you are adding a post-addition that has been made with a contaminated water source or your aseptic techniques are bad. It's very rare to get a phage in a shake flask, with an isolated system, you'd have to have one serious contamination, and if thats the case, you'll need to hire someone to come in and clean your facility (Critical Cleaning here on the East Coast can do peroxide bombing). Before you go that far check that the antibiotics and the iptg you are making is made with sterile water. Autoclave a bottle of your DI water and use that in the hood to make any additions to your flasks. If you're flask is autoclaved properly, you've autoclaved your media and autoclaved your water for making posts, then you should be able to grow in a shake with no problem.

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Rumo
Have you sorted out your problem?
We experience the same thing in our lab and it is so fustrating. Its been happening on and off for the last year or so, no rhyme or reason to it. When the cells reach an OD 0.4 - 0.6, sometimes before the addition of IPTG, they start to lyse and the OD drops dramatically. Sometimes if I inoculate 6 flasks from the same o/n 3 might grow while the others lyse.
Our cleaning procedure
After using the flasks we soak them in virkon solution o/n then rinse and send them for washing. Then we rinse again before putting in new LB and autoclaving

Any ideas or thoughts would be greatly appreciated

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Hi all,

Hey Rumo,did you find the reason of your problem? (Hopefully yes ) I've having the same problem here in our lab . The O/N culture grows nice, even after put it into the big flasks but after the inducion with IPTG the bugs grow until an OD ~1.3 and after that the OD start to decreases and the culture becomes more and mores clear and with a huge amount of foam. Its less commom to happen but sometimes my o/n culture dies. I've checked several things that could be the problem (media, flasks, antibiotics,IPTG,competent cells...) and no one seems to be the reason.And something that is really weird its that sometimes I got expression and sometimes not in the same flask, using the same fresh colony to prepare the O/N culture.
If u know the reason , please, let us know whats that thing and how did u verified it.

thanks =)

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Hi!

Some basic solutions for such situations you may find on www.phageconsultants.com - in section with publications. If the problem is more serious I advice to seek for help.
Best regards
Marcin Los

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