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Contamination in cell cultures


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#1 helpme

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Posted 16 March 2010 - 05:39 PM

Any help would be greatly appreciated. This has been a problem for about 1 or 2 years now and we are getting desperate! To start off, I'll give everyone some background. I didn't join the lab until 2 months ago (after all this happened) so I will try my best to explain.

We ordered a rare primate cell line from Coriell and went about culturing it. However, a strange hair-like particle started appearing in the media of all the cells (293T, CHO, etc). At first, we didn't suspect that it was a contamination problem but it was consistently found in all our cultures. Finally, after some investigating, our lab contacted Coriell about this problem. They claim that they didn't see anything in their cultures and agreed to send us another flask, free of charge. The older lab members have told me that as soon as they received the flask, they put it under the microscope before unsealing it and saw the same exact thing. Again, Coriell didn't acknowledge the problem.

To completely decontaminate the tissue culture room, EVERYTHING was thrown out and the hood was dismantled for disinfection. There was a theory that this thing was in the incubator so we even got an elaborate new incubator that cleans itself and threw the old one away. We put in hepa filters all throughout the lab. Everything was replaced and put back. The first day that the tissue culture room was up and running, we found it on the outside of the flask. Before long, it started appearing in the media again.

I started doing work in the tissue culture room after it was decontaminated. I found it in all my cells, especially in the ones that I am performing antibiotic selections on. I don't see more than 1-3 in 1 flask so it doesn't seem like it's getting wildly out of control but it is still stressful because I don't know what effect it has on my cells. I have done a lot of cell culture work before joining this lab so I know that it isn't my technique. I've started using a pen/strep/antimycotic solution in my media and I don't see it before I passage but as soon as my cells are split and new media is put on them, I see one in there. However, it disappears before it is time to passage again. I wipe everything down with Vikron and then 70% ethanol before putting anything back in the incubator. I have 6 cell lines going and I will have 5 more soon.

We really want to get rid of this problem and we want to find out what it is exactly. I have attached a few pictures of this thing. Please help us pinpoint what it is!! Our last resort is to hire a company to come out and decontaminate the whole room using a hydrogen peroxide vapor generator. That will be REALLY expensive so obviously we want to get rid of this with a cheaper solution.

Using our brand new awesome microscope and camera, I've taken some pictures. Any input would be greatly appreciated!!! Please help us figure out what this is. Sorry for the long post but I thought it would be best to include as much information as possible. Thanks!

Attached Thumbnails

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Edited by helpme, 16 March 2010 - 05:40 PM.


#2 Clare

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Posted 17 March 2010 - 03:12 AM

Sorry to hear about your issues B)

If this 'thing' doesn't seem to be growing, I suspect it's just crap in your cultures (ie: a bit of plastic or packing etc from something you use in tissue culture). It doesn't look like contamination to me. I have seen this before and haven't been concerned.

Clare

Edited by Clare, 17 March 2010 - 03:13 AM.


#3 jakatta70

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Posted 17 March 2010 - 05:12 AM

Hi,

I completely concur with Clare. This is only a piece of plastic from the wrapping which either the pipettes or flasks etc have been in. I wouldn't worry about it. We see it regularly in our cell lines and it does not seem to have any disastrous effect on growth, apoptosis, necrosis etc.

#4 hmohamma

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Posted 23 March 2010 - 07:54 AM

Hi,

As other said I think it is not contamination too. I also think it might be from the serum proteins that you are using. You may centrifuge the serum and take the supernatant also could filter it. hope it helps.

Regards

H

#5 leelee

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Posted 23 March 2010 - 05:53 PM

From your pictures it doesn't appear to be in the same plane of focus as your cells, could it actually be on the outside of the flask? When you are focused on one, try brushing it away by wiping the underside of the flask.
One of our honours students had this exact same looking thing, and it was just on the outside and we could wipe it away.

#6 DRT

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Posted 25 March 2010 - 12:58 AM

Nice images.

What is this new microscope/camera set up you have?

#7 helpme

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Posted 25 March 2010 - 08:13 PM

Hi everyone. Thanks so much for your help!

It's reassuring to know that people have seen something similar. The problem is under control now but we see it from time to time. We thought it was some kind of fungus because it disappears after we use fungicide. It floats in the media and can sometimes be found on the outside of the flask (I can wipe it off). It seems to be more prominent when I'm doing antibiotic selections and "sticks" to dead floating cells. We also filter all our media before we use it to eliminate the possibility that it might just be precipitation of some component.

It isn't causing major problems with my experiments so I will just keep going. Maybe we'll try to switch pipettes? Or try a different brand of flasks? Thanks again for all your helpful comments. I feel better about it now.

The pictures were taken with our new Zeiss Axiovert microscope and monochrome ccd camera. Thanks for noticing! :lol:

#8 helpme

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Posted 25 March 2010 - 08:19 PM

By the way, some of the pictures were actually taken after I passaged the cells and noticed that the "thing" was still in the empty flask. That's probably why it looks like it is out of plane. Sorry for the confusion.

#9 Chantelle

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Posted 26 March 2010 - 07:35 AM

By the way, some of the pictures were actually taken after I passaged the cells and noticed that the "thing" was still in the empty flask. That's probably why it looks like it is out of plane. Sorry for the confusion.


the picture shows artifact more than fungus thread, it might be a cotton fiber?
but if you have suspecious then you can sterilize your lab with formalin ( like operation theater)

one more thing Fungus if grow it will fill your flask not few threads

I'd like to suggest to cultivate your cells with this thing in fungus media and see what can you get ?
Good luck

N.

#10 klwenk

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Posted 23 June 2010 - 07:23 PM

I started doing work in the tissue culture room after it was decontaminated. I found it in all my cells, especially in the ones that I am performing antibiotic selections on. I don't see more than 1-3 in 1 flask so it doesn't seem like it's getting wildly out of control but it is still stressful because I don't know what effect it has on my cells. I have done a lot of cell culture work before joining this lab so I know that it isn't my technique. I've started using a pen/strep/antimycotic solution in my media and I don't see it before I passage but as soon as my cells are split and new media is put on them, I see one in there. However, it disappears before it is time to passage again. I wipe everything down with Vikron and then 70% ethanol before putting anything back in the incubator. I have 6 cell lines going and I will have 5 more soon.


After looking at your pictures i'm SO relieved because i have been seeing those things in my cells too, especially after seeding them in dishes for antibiotic selection (presumably because the cells have gone through more plastic surfaces, from flask to tube then to plate). So I keep throwing my cells away once i see those things and it's painful :'( One thing that worries me so much is that they are either floating or stuck to the upper plane of the flask (the 'ceiling' where the cells or media never reaches), and i'm new to cell culture work so i have no confidence in my techniques at all..been losing sleep over this for months :angry: Does anyone have any suggestion to help prevent or at least slow down the rate of this buildup? thanks so much for all the help and posts!




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