E.Coli culture contamination 2 - (Oct/11/2006 )
Hello everybody,
In my lab we are having problems of growth of E.Coli cultures, they start to growth but at 0.3-0.6 the OD starts to decrease and the culture apparently dies.
Does anybody know what kind of contamination could it be (a part from phages, I am pretty sure that this is not the problem, cause we sterilized all at the UV) given that the media, antibiotics are all ok?
Thanks!!
Roman
You could have bad cells or a toxic protein. Are you trying to express a recombinant protein? Some vectors are leaky and will express low levels of the protein without being induced. I have had the same problem in the past and it turned out to be a leaky vector.
Yes, I express recombinant proteins, but the constructs that are giving me problems now grew and expressed perfectly in the past... It could be a problem with the cells, altought the transfomations run apparently well?
You havent specified transformed/untransformed.
did you plate and saw what kind of colonies you get?
e. colis are typcal
In my lab we are having problems of growth of E.Coli cultures, they start to growth but at 0.3-0.6 the OD starts to decrease and the culture apparently dies.
Does anybody know what kind of contamination could it be (a part from phages, I am pretty sure that this is not the problem, cause we sterilized all at the UV) given that the media, antibiotics are all ok?
Thanks!!
Roman
could we have some more info pls,
which strain?
which growth media?
which growth conditions?
are they transformants? and if yes what have they been transformed with?
Cheers
Yes of course,
the strain was BL21 (DE3) and we have had problems with both Kanr and Ampr contructs.
We are pretty sure that the transformations grow ok; and the liquid cultures, where we are having the problems were LB; it is not a problem of degradation of the media though, because the death of cultures also ocurred with new media. I am sure it is a contamination but we have not found where is the origin
(P.D- Since two weeks, it seems that the incidence of the problem is disappearing but we don't know exactly why, cause essentially we work the same)
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.
It happens throughout the lab in different strains, BL21, Rosetta, Rosetta2
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
Hello,
We are still with the problem, that sometimes comes and sometimes goes, but now we have more information, because we asked for contamination analysis of the lab and now we just have the results. It seems that we have a lot of contamination from staphylococcus cogulasa (gram + bacillus), but the focus of the problem is in the air conducts, so we are going to do an exhaustive cleaning. I am not sure that the bacillus affect the E.Coli growth and even could induce the lysis of E.coli. Before, we thougth thta what we had eas a proble of phages, but we will see if the cleanings are efficient and I will tell you whether we overcome the problem or not
See you
We are still with the problem, that sometimes comes and sometimes goes, but now we have more information, because we asked for contamination analysis of the lab and now we just have the results. It seems that we have a lot of contamination from staphylococcus cogulasa (gram + bacillus), but the focus of the problem is in the air conducts, so we are going to do an exhaustive cleaning. I am not sure that the bacillus affect the E.Coli growth and even could induce the lysis of E.coli. Before, we thougth thta what we had eas a proble of phages, but we will see if the cleanings are efficient and I will tell you whether we overcome the problem or not
See you
Hi Rumo,
We 've having the same problem as you and I found bacteriophages in our lab. Its very inconsistent since it doesnt happens always so we are trying to look what is the major focus of this contmination. You should be carefull with the pipetes. I know that at least the set of pipetes that I've using are contaminated. You can also verify is you have contamination of your glicerol stocks. The best think you can do is spread an ammount of the glicerol stock into an agar plate+ antibiotic and leave it grow until cover all the plate. If you find transparent spots into the plate that means that you have phage.
We will perform an assay soon to check the other glicerol stocks . If it works i will let you know
Cheers