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helpme 03-13-2010 03:25 AM

Another contimation problem
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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!

sweetsunil21 05-18-2010 09:35 AM

Re: Another contimation problem
i posted already a reply but its not appearing

i said it may be in media powder.
i too got it many times but not continuously.

Bonnie 05-18-2010 09:41 PM

Re: Another contimation problem
I've seen that kind of thing many times in my media. I can't identify it, but I don't believe that it's anything alive or any kind of contamination. It doesn't reproduce or spread, and I've never known it to cause any change to my cells.

I suspect it's some kind of precipitate from the media, since I think I've seen it in plates where I hadn't plated any cells yet.

Bonnie 05-18-2010 11:13 PM

Re: Another contimation problem
Actually, I just went and looked at some plates, and I can see strands that look like your pictures in dry, sterile Corning cell culture plates that I just took out of the package. When I add sterile PBS to the plates, the strands float around in it just like what you're describing.

So check your plates before you add anything to them to make sure, but the strands may just be inert bits of plastic leftover from the manufacturing process.

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