Help me brainstorm an explanation for this cell culture contamination
Posted 11 July 2011 - 02:24 PM
So here's the scoop.
A couple of weeks ago, I thawed a vial of NIH 3t3 mouse fibroblast cells (I was not the one who made the freezedown stock, nor am I sure if this was a commercially obtained vial or one that was previously frozen in our lab). Last Wednesday (7/6) I split them for the third time. I checked on them every day until the weekend, they were growing slowly, but I chalked it up to the fact that I did a 1:10 split and the cells were about ~75% confluent when I had split them. There was no indication of bacteria in the flasks when I checked them. Today, I did find a contamination. They were rod-shaped, black, motile and growing in small groups together. The cells themselves were stretched very thin and did not look happy (yes, I know that sounds so scientific). However, this contamination was definitely still in it's infancy. I know its possible my sterile technique isn't perfect, but I did everything by the book so to speak. Also, my older cell line (currently in its 9th passage), which was split the same day as this, was totally confluent and contamination free this morning. Of course, I got rid of these cells, but the fact that I needed to bothers me. And when I think back on the history of these cells from the beginning, I realize their behavior isn't consistent with what I know about the NIH 3t3s (ie. that they grow very quickly). From the moment they were thawed, these cells were very slow growers and looked slightly better than the way they did today with regard to their shape, but not the way my older cell's looked. Do any of you believe that its possible that these cells were contaminated to begin with and it just took awhile to appear? I know, ~2 weeks sounds like a stretch), but the fact that it also took 5 days for a small amount of bacteria to show up has got to mean something. Also, just to let you guys know some other details, my media, trypsin, and PBS are definitely fine given that the other flask doesn't have any problems, and our incubators were cleaned last week as well and no one else had contaminated cells, so its definitely not the incubator.
I would really appreciate it if people would give their input. Contamination really bugs me.
Posted 11 July 2011 - 03:57 PM
It is entirely possible that the stock was frozen down with a low level contamination, they can take a while to show.
Posted 12 July 2011 - 01:22 AM
if u want to make sure your media is fine you can incubate it for some days and see if some contamination appears after some days.
otherwise throw out the stock. indeed, happy nih3t3 cells will grow like hell in healthy conditions.
Posted 12 July 2011 - 04:17 AM
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