persistant bacterial contamination, causes and solutions - (Sep/23/2007 )
Hello all,
I am facing what has to be the worse string of bacterial contamination in my years of science. I am culturing both mouse and human ES cells, and in the past 2 months I have had persistent contamination, mostly in the mouse cells. It appears mostly as rod shaped particles that exhibit robust movement within the culture, along with cloudy yellow media. To the best of my knowledge and experience it appears to be bacterial in nature but any second opinions on that would also be helpful.
Most of the cultures show contamination when undergoing differentiation, on gelatin coated 24 well plates. The contamination appears randomly in the plates, sometimes after 5-7 days in culture, and may strike 1-2 wells or 8-10 wells at a time. I have to feed them every day so the expansion goes from possibly visible but not obvious to obvious contamination within 18-24 hours. These culture are being grown in a pen strep containing media and still the bacteria are growing.
I have tested the medias used, filtered them and then just incubated them along with the growth factors and other reagents that come in contact with the cells. So far nothing has popped up on these screens. This has led me to believe that the cells themselves may be harboring the contamination. The mouse ES cells are being transfected using electroporation, but the infection has appeared only very rarely in the expansion cultures. Initially the cultures are selected with g418 but after selection are cultured in just pen strep containing media.
My questions are thus:
1- Does sporadic bacterial contamination, in the time frames involved (amywhere from 2 days to 10 days in culture) suggest the source of the contamination is the cells themselves?
2- If this contamination is not held in check with pen strep containing media is there another antibiotic addition that might help curtail this infection, or is even thinking about trying to save cultures that appear to have a pen strep reistant strain of bacteria a fools errand and could potentially cause more prolems down the road?
3 Anything else pop out from my description as a concern or any other suggestions.
Thanks for the help. I am 6 months into a new post doc and this is really starting to frustrate me and my new boss, not to mention that I have a meeting coming up where I have to present a poster on the data that these cultures should be providing, and time is running out.
Thanks again
Sean Kearns Ph.D
I am by no stretch of the imagination an expert on cell culture- but heres what I think:
It sounds like bacterial contamination to me. Seeing as you have tested your media and that also does not seem to be the source, I would suspect that something else you are using is contaminated. For example, do you use re-usable glass pipettes?? It is possible that they or something else you use are not being sterilised properly??
Firstly I would completely decontaminate and clean your hood , I don't know what cleaning products you use, but I would ethanol then bleach then rinse bleach off with sterile water then ethanol again. Also I would clean and decontaminate your incubator. And MOST importantly, ensure you are using correct (and strict) aseptic technique when performing cell culture (which I'm sure you are 
)
Has anybody else used those same cell stocks and had the same problems?? To me it seems highly unlikely that your cell stocks are the source of the contamination, you could check this by thawing a vial of your stock into media WITHOUT antibiotics and leaving it in the (now clean) incubator, checking daily. As far as trying to rescue the cells that are contaminated- if you can avoid going down that road I would. From what I understand, infection has the potential to cause a change in the cells (eg cell behaviour) and this may affect your results. It is best practice to toss the cells and start again.
Do you ever see contamination prior to electroporation?? Is your electroporator used for any bacterial work?? This was something that jumped out at me in your post.
I wish you every luck in isolating your contamination source!!
Another thing.... have you tested your trypsin and PBS for contamination??
Thanks for the responses. As far as those scenarios:
- There has been some sporadic contamination in other user cultures, but nothing as prolonged and repetitive as mine. We have cleaned and autoclaved out our incubators and hoods recently.
-The electroporater is hardly used at all, I am the only one, and we use sterile one use cuvettes. I am just wondering if contamination is getting in through the vector solution (vector plus TE buffer)
-I tested the hanks we use as a wash and did not see anything. I have not tested the trypsin, but all users use frozen aliquots from the same source so if I was getting it from the trypsin I suspect others would be having more problems.
- I am not the only one using the mouse cells, but we each keep our own stocks of the cells and so far mine are the only ones showing contamination. I am the only one doing electroporation however of the cells.
Sometimes water bath could be a source of contamination as well.
Typically contamination is introduced through accidental touching of pipets to a contaminated surface.
Hi,
well I also experienced something similar recently.....I would suggest decontaminating your incubator, if need be, clorox it, thoroughly sterilizing your shelves, cleaning the water bath....there could be several sources, do you perform your cell culturing in a "clean room," is the air under your hood being filtered properly....have you tested for mycoplasma contamination?if you are unable to trace the source and you were keeping frozen stocks, they might also have been contaminated as well. We have started autoclaving our PBS after every usage...you may want to start fresh with new cell stocks if you can.....change out your flasks, sterilize everything, and spray everything with methanol, decontaminate your hood, and just be anal for the next couple of weeks and see how that pans out.
well as it's repetitive, it comes from the medium or pbs (easy to change) but also from serum or others adjuvants you use. If you can, filter on 0.22µm filter then uv or gamma sterilize all expensive reagents.
The point is that all things should be changed on the same time, as well the incubator should be cleaned, and sterilized (many have a 95° cycle which sterilize). The water introduced in autoclave should be milliQ one in order to minimize contamination potential.
Check the pipettors, if they are autoclavable, do so. If not, take apart every piece you can with out damaging and clean with 70%Etoh.
Include the large volume dispenser.
Are you using disposable filters? The reusable ones can give you trouble if not properly handled.
Is anyone using the hood before you? A month ago I caught an _____ doing bacterial cultures inside the cell culture hood. 
Thankfully He had not done this before so no damage was done.
well I also experienced something similar recently.....I would suggest decontaminating your incubator, if need be, clorox it, thoroughly sterilizing your shelves, cleaning the water bath....there could be several sources, do you perform your cell culturing in a "clean room," is the air under your hood being filtered properly....have you tested for mycoplasma contamination?if you are unable to trace the source and you were keeping frozen stocks, they might also have been contaminated as well. We have started autoclaving our PBS after every usage...you may want to start fresh with new cell stocks if you can.....change out your flasks, sterilize everything, and spray everything with methanol, decontaminate your hood, and just be anal for the next couple of weeks and see how that pans out.
Personally I have suspected the frozen stocks for a little while. The problem is that the contamination does not appear in all the wells from each cell type or treatment. At first I thought it might be the growth factors, because a lot of the contaminatied wells were getting that treatment, but a lot that have been treated with GF have been ok. In addition media with growth factor placed in the incubator has not shown any signs of infection after 3 days so I am doubting the media or other components. Also all the media is filtered prior to use. Then I started to suspect one of the transfected cell lines, since it was showing up more contaminated than the controls, but once again it was not in all of the transfected cell wells.
At this point I have cleaned both the hood and the incubator. The water bath is next but we have this blue water treatment antimicrobial and anything in the bath is sprayed with ethanol before entering the hod. I am getting ready to thaw out a early passage cell aliquot and redo the transfection, hopefully that culture will remain clean, but if not then I am really at a loss.