8 replies to this topic

[CAT]

Hi guys,

  I need help to identify what kind of contamination happened to my cell culture. I have HEK293 just thawed from liquid nitrogen and split just once. Then I found this contamination:

When I swirled around the culture plate, a puff of powder-like or dust-like things were blew up from the bottom of culture plate and then sink down. It could be observed directly by eyes. This cell culture contaminator feels and looks a lot like the bacteria culture flask: when you swirl the bacterial culture flask, the cultured bacteria are blasted up.

1) medium is not turbid, still clear and  with normal red or purple color (PH normal). (so , it is not bacteria contamination?)
2) cells are still attached well. no obvious cell debris floating in the medium.  Checking under light microscopy, cells have normal morphology.
3) The contaminators did not have the shapes of thread, rod or branches. Anti-fungus agent not help either.

So, anyone has any ideas? Many thanks for your time.

[scolix]

Is it something from the media?

[CAT]

not likely!
It was fine a day before and happened later. Definitely not from Media.

[scolix]

if its contamination, better throw it out before other culture plates get contaminated.

I haven't encountered some thing like this before.

[bob1]

It wasn't just cell debris/dead cells?  Could you post a picture?

[CAT]

I thought of that either. But is it just weird that such amount of cells just died in one night? Sorry no picture taken so far...

[bob1]

293 can be quite weakly attached and if recently thawed you may see a lot of debris from cells that didn't survive the freeze/thaw.

[CAT]

Thanks, bob1.  
It is unlikely due to frequently freeze/thaw cylce 'cause this dust-like problem came back again to the newly spit cells. This is what I did:  wash with PBS twice-trypsinize- add serum-containing growth medium to stop trypsin- then directly seed to a new flask. With help, now, I could upload some pics, taken with 10X objective.  One of them (forgot which one, ) is off focused and trying to take a snap at those floating unknown, Please suggest. Thanks!

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[bob1]

Hmm, it's a little hard to tell from the pictures, but I would have said that you may well have bacterial (or perhaps yeast) contamination.  If it is yeast, on a 20 or 40x objective you should be able to see the classic budding structure of the yeast cells.  Bacteria usually appear as a shimmering cloudyness in the medium and you may not be able to see the contamination with the naked eye until it is quite heavily grown.