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Help me brainstorm an explanation for this cell culture contamination - Cell Culture, contamination, NIH 3t3 (Jul/11/2011 )

Hey everyone,

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.


DO you have antibiotics in your medium?

It is entirely possible that the stock was frozen down with a low level contamination, they can take a while to show.


sounds like the stocks are contaminated.
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.


No, my mentor doesn't like to use antibiotics in our media--he says they just cover them up and appear at crucial times such as plating before a protein extraction and then they appear contaminated when you need them. I'll take your advice about incubating the media, but if its the contaminated thing, I would assume my cells that are still in the incubator will become contaminated in a couple of days.


Sounds like a classic case of contaminated stock--if it was your media all your flasks would be toast (especially since you don't use antibiotic). If that ever happens with a really really really important or expensive vial and you have no back up you could always try a FBS underlay. My old mentor showed me how to do it. Tryp your cells and transfer to a 50 conical tube (total volume of no more than 30mL). Then use some nice think sterile FBS and very carefully and slowly add it to the bottom of the tube (about 20mL) so that there are two separate layers. Then you just centrifuge at like 100g for 4min and the cells will pellet but the bacteria (if they are not attached or intracellular) will be stuck at the interface. You just aspirate everything (I usually suck down to about 15ml left and then switch to a new aspirating pipette just to be safe. If you do that a couple of times you should have a nice clean culture again--just to be safe I will sometimes still grow with a little penn/strep for a couple of passages but I have never had contamination come back. Good Luck!


That proceedure won't get rid of intra-cellular bacteria.


True, it won't get rid of intracellular bacteria, but if you have myco contamination you probably wouldn't even know it. Usually if you can see the contamination it isn't intracellular. That is also why using antibiotics isn't a good idea because many intracellular pathogens aren't killed by them (ie mycobacterium).


You do have to keep in mind though, that perhaps the infection may have had an effect on the behaviour and properties of your cells, so even if you can rescue them, they may never be the same...