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Is this contamination or cell death?


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#1 Johannes

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Posted 10 December 2012 - 04:02 PM

Hi Guys,

I just recently started cell culture and got my first cells in and already something seems fishy.
I have 2 celllines, MRC-5 and BHK-21. I grow both of them in the same medium (DMEM high glucose +10% FBS).
They both grow, but in the BHK-21 flasks I see a lot of cells detaching and swimming in the medium. And after some time these things here show up:

bhk-21-02.jpg
bhk-21-03.jpg
bhk-21-04.jpg

So far I took some of the supernatant and put it on TSA and SAB agar plates and incubated them at 37°. After 5 days in the incubator there is nothing growing on them yet. Can anyone of you more experienced culture users tell me what I am dealing with here?

Thank you very much in advance.

Attached Thumbnails

  • bhk-21-02.jpg
  • bhk-21-03.jpg
  • bhk-21-04.jpg


#2 bob1

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Posted 10 December 2012 - 06:41 PM

They look like senescent cells, but it is a little hard to tell from the pictures.

#3 Johannes

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Posted 10 December 2012 - 11:55 PM

I just found this on wikipedia, it looks quite like the things I observe under the microscope:

http://en.wikipedia....E_syncytium.jpg

I might have caught a virus then ;-(

#4 bob1

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Posted 11 December 2012 - 01:00 AM

I suppose it is possible, but the number of viruses that cause syncytia is fairly low and you would probably have to cough directly into the flask to get the cells infected like that.

#5 fraffly

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Posted 12 December 2012 - 06:20 AM

Doesn't syncytia occur quite commonly in some cell lines ? I've observed similar looking cells multiple times in different cell lines in different labs. I think some retroviruses can cause that too.

There seem to be quite a few multinucleate cells (if they are syncytia) around in those pictures though, might have something to do with the selection pressure due to passaging behaviour. In my experience they adhere much stronger to the plastic than the "normal" cells, so they tend to accumulate when the same flask is used for several passages.
Changing flasks more often helps to reduce their number.
Also, since the normal cells usually detach faster than the syncytia, it's easy to time the trypsinization and just passage the normal cells.

#6 bob1

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Posted 12 December 2012 - 11:53 AM

What you may well be mistaking for syncytia are more likely to be senescent cells. These are cells that have permanently exited the cell cycle and are typically large, multi-nucleate, often vacuolated and will accumulate in a given population of cells over time.

#7 Johannes

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Posted 20 December 2012 - 07:25 PM

Hey guys, thanks for your ideas.
@fraffly: I am not reusing flasks, i grow cells on disposable plasicware.

I grew the cells for a few days on coverslips and stained them with crystal violet.

I am sorry for the poor quality pictures, but we do not have a proper camera setup on the microscope, so I am using a regular digital cam and hold it to the eyepiece ;-)

Attached Thumbnails

  • bhk-21-crystal-violet-01.jpg
  • bhk-21-crystal-violet-02.jpg
  • bhk-21-crystal-violet-03.jpg


#8 bob1

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Posted 21 December 2012 - 12:02 AM

They do look quite like syncytia, but I still think they are senescent - note the vacuoles that are visible in some of the pictures.

Your pics are quite good for hand-held down the microscope. I have definitely seen worse on here from people who have proper microscope cameras.




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