We are facing a problem in different groups in our institute: when we grow E. coli (DH5a) overnight in liquid LB, we have:
- no growth at all
- sometimes, some tubes show cell growth, some don't
We see that with different antibiotics, and lately, someone even tried with LB without antibiotics, and nothing grew!
This is going on since January, and we really don't see what is going on, the only common factor is water (but this same water was used for solid media, and cells grow well on plates!)
Have you ever faced such problems?
Dear Noémie, in my opinion, the reason is viability of DH5a. Too much freeze-thaw can affect viability of DH5a cells.
Best regards.
It grows on plates, and then the colony does not grow in liquid. And from a same plate, we pick 10 colonies, and then not all of them grow...
We think that there is a problem with the oxygenation, but what exactly....
Dear Noémie, maybe you can check the growth of DH5a colonies in other media e.g. tryptic soy broth or brain heart infusion broth. Maybe there is a problem in your LB.
good luck
If LB without antibiotics is not supporting the growth of your bacteria then you have a systemic problem of some sort. It could be the water or the batch of reagents used to make the LB (although if the LB plates are working that might suggest otherwise). It could be someone in the kitchen making your media is misreading the recipe. Its could be the tubes or flasks you are using. If you are using glass tubes or flasks for example that get washed. perhaps they are not washing out the detergent adequately, or a batch of disposable glass tubes is similarly dirty.
From my experience MJ Benedik's idea is likely the right one: It's not the media but the growth tubes. Suggest: Put 20-30ml of the same liquid LB into a fresh petri plate and inoculate it and let it sit overnight. See if it grows reliably. A great geneticist I knew would allways wash her own glassware before an important experiment. Residual detergent is very toxic to E. coli, she rinsed it 7x.
This strain of E. coli is dlacZ Delta M15 Delta(lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1. Try supplementing the LB broth with the vitamin thiamine (10 microgram/ml) and arginine (10 milligram/ml).
It's weird. Maybe try to wash the tubes or flasks thoroughly and then sterilize them.
hi Noémie,
Is it only the DH5a which is giving such problem or the other cultures are also showing similar effect. If so then most likely it is because of the bacteriophage contamination.
we have faced a similar problem with cultures not growing at all. turned out to be bacteriophage contamination.
You need to properly wash all your utensils (flasks/tubes/ beakers/bottles/etc.) and get them autoclaved. hope that will work for you.
Yes Bharat's answer makes sense. If phage contamination is happening you might notice if colonies on the plates have a nibbled irregular appearence and are maybe translucent in places.
Thanks for l these answers. The most probable hypothesis is phage contamination. Since we often work without a hood for miniprep cultures, it makes sense.
It is quite astonishing that it happens to different floors of the same building almost at the same time... Does anyone know how bacteriophages contaminations can occur? Is it something staying in the lab for long?
Of course, with better lab practices we'll get rid of this, but I am just wondering...
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