Contamination issue in cell culture - (Nov/16/2004 )

Hi frusted PhD (shouldnt we all be named like that???)

We've had the same problem for weeks and eventually we found out that the PBS was the problem(pH or something else we dont know) so we started to wash our cells with plain media instead of PBS and cells survived. :-) even under low serum conditions. Now we only use premade PBS from GIBCO to wash our cells. It might sound absurd but we checked over and over and the PBS must have been the cause of this all. Not sure if it's applicable to your case but it might help you.

Good luck

another frustated PhD

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This contamination has been an aweful problem for our lab since the beginning of the year and has virtually eliminated our ability to perform informative tissue culture experiments. We work with several immortal cell lines (i.e. HeLa, 3T3, Cos, SkBr3, P19, etc.) and have found this contamination present in every circumstance. As described by others, its very small, slow-growing, doesn't change medium pH, no turbididty, doesn't kill cells but makes them sick. These bugs seem to live on or in the cells. We have also noticed that serum starvation causes the cells to detach. We are a cell signaling lab and find that many signaling proteins are constitutively active in the presence of this contamination. We sent three samples to our diagnostic laboratory and two times, they said there wasn't any contamination and one time we got three species back, so we were never able to make a positive I.D. on what the organism is. We find it to be resistent to Pen/strep, neomycin, G418, etc. The only antibiotic we have had any success at all with is Cipro. Scarey. Given that we couldn't come up with a common reagent that was contaminated (given the many different medias and serum we were using),and that sterile filtering was of no use, we suspected that the contamination was environmental. However, I began testing the serum, FBS and newborn CS. Suprisingly, everything I tested from Invitrogen, Cellgro, Atlanta Biological and Gemini was coming up contaminated. This seemed highly suspicious given the different sources of the sera and that they are coming (apparently) from different processing plants. Recently, I tried some gamma-irradiated FBS from Invitrogen and the results were amazing. I brought up 3 new cell lines from the ATCC and there was absolutely no signs of contamination. Furthermore, our functional assays showed proper regulation of signaling molecules. We have since purchased large lots of irradiated FBS and CS and hope this will be the end to the problems. I really worry, however, that this is a widespread problem in the scientific community and that it is escaping the notice of many laboratories. Invitrogen (where we usually purchase serum from) claims that there have been no complaints about this contamination. I would encourage anyone with this contamination to test the serum (beware, it takes at least 2 weeks- the bugs don't grow well without the cells) and then complain to the company supplying the serum if you find a problem.

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We too are facing the contamination problem in our primary culture. Eventhough we are trying to follow the most stringest methods and include double conc of antibac anti fungal things still we are getting these spherical demons in the plate.(Attached picture!)
Can anybody tell me what it is? I guess it being fungal may be yeast! Upon addition of fungizone the sphere gets dismantled in to small fibres all over the plate and over time again accumalate to give such spheres!!

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I'll say it looks more fungal then yeast. semantics
It seems to have formed a nice (or not so nice) fungal body.

I am not a fungus expert, but I can safely say that is not S.pombe, S. japonicous, S.octosporus, S. cerevisiae, S. paradoxus or even C. albican.

Good luck to you guys. I sure am glad that I don't really work on vertebrate cells... Your horror stories are truly scary. It kind of put things into perspective when compared to my 'slow' growing yeast.

EDIT : Just had a strange idea, I am not sure about the magnification, but could this actually be a wall deficient bacteria, something with a fungoidal form? Erm... there is nothing here to see here... just a fool with a microphone.

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Hai,

Same problem with my primary culture.I am doing my routine culture also in the same hood where I do my primary culture. Now i have noticed this contamination both in my primary and routine culture.i can throw out my routine culture but my primary culture cells are precious as I got it for the first time after trying with too many tissues. Now when the procedure got standardised, these devils came to get the hell out of me.So please can any one give a suggestion to get rid of these conatminants so that I can maintain my primary cells

Thank you

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Hello,

I too have encountered this contaminants. In fact, they costed me a chapter in my PhD thesis because I couldn't get my cell cultures going, all thanks to these little bastards. This was back in 2001-2002. I wonder why this problem is only noticed recently (after 2000). Nothing I did could get rid of them. I am convinced that the contamination did not come with your reagents.......PBS, FCS, media, water or bath water. It is in the air! I did a simple experiment. I thoroughly cleaned out my incubator with paraformaldehyde and Virkon and then asked for a flask of clean cells from another lab, which is located far away. I put the flask in a clean container during transport. All I did was loosened the cap and put the flask inside my incubator. Next day, it was teeming with those little creatures.

I have filtered all my media and reagents with 0.1 uM filters, bought several batches of cells from ATCC and FCS from Gibco and Hyclone, put filters in the air duct to my lab etc etc but to no avail. Later, when I moved to another institute to do my postdoc, I discovered that the whole institute had this contamination and nobody realized it! They never look closely at their cultures! And the whole liq N2 tank contains tubes after tubes of contaminated cells.

In addition to all the antibiotics listed by others in the previous posts, these bugs are also resistant to vancomycin. I noticed that certain cells are more susceptible to these bugs. During my PhD. I was working with Y1 adrenal cells. These are absolutely sensitive to the bugs. They contract forming threadlike processes and eventually disintegrate into a thousand pieces. Mesenchymal stem cells also seem to be sensitive. However, cells like HeLa, Cos7, and 293FT seem to be able to tolerate. CD34+ stem cells seem to be completely immune, maybe because they can differentiate into the right kind of cells and counterattack the bugs.

We should do something about this bug. It is ruining many careers and degrees. As someone mentioned in a previous post, it can be the subject of an interesting paper and whoever discovers a solution for it would potentially become very rich!

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From the past one month,there has been contamination in HEK293 cell line which i use.I tried using fresh stock,but everytime there is same problem.The cells start detaching & floating in media after 2-3 days & after 10-15 days,the media becomes cloudy. Im new to this field & im really very worried about this.Can someone give me any suggestions?????
Is it okay to put the pipette directly in the media flask or should we pour media in a falcon tube & then pipette it?Can it cause contamination?
If we do not split the cells when they are fully confluent & keep them for some days,can it cause contamination?
Plz give me some tips.....

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you can put sterile (individually packaged) pipettes (2, 5, 10 mL) but not tips (10 µL, 100 µL).
when you open your individual package, do not tear the package with the pipette, but pull the two folders where it is written, unless you might touch the outside of the torn paper with the extremity of your pipette while pulling it outside (do you see what I mean?)

Do not lay down the bottles when you store them. no liquid should touch the cover (the cork).

when you work under the hood, wash your hand with detergent and then with alcool.
Keep in mind that gloves are not sterile, it's only to protect you.
always let a the lid on the bottle. Do not let an open bottle under the hood. you don't need to screw it each time, just let it on the top.

do not work too close to the opening. you must let around 10 cm to the opening.

if you need to pipet small volumes with a tip, never do it in a bottle, you have to transfer to a smaller volume so that the micropipette never enter the bottle.
clean all what enter under the hood with ethanol.

avoid to pipet too quickly because you would make some aerosols, that could contain contaminating particles.

Now throw away all your medium, antibiotic, serum aliquotes, take fresh cells and try again.
are you the only one that has contamination? maybe the incubator should be cleaned also.
there are some products to clean it (some are even autoclavable), you can autoclave the shelves.
Clean the hood also, especially under the surface where you work.

you should not keep contaminated cells in the incubator, but over confluent cells are not contaminated and this will not cause contamination.

do you work with petri dishes of flasks?
with petri dishes you will have more easily contaminations, especially if the incubator is over crowded, because persons caring less than you could move your plates to get their plates, and if you are not carefull (no hand wash...) you can split some medium to the opening, and contaminate the medium.

OK good luck

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very nice answere - to print and hand-out for beginners

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yeah .. Mods Pin this.

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