Black dots-ghost cells or contamination? Please help - (Jan/26/2015 )

So I have been working with PC12 cells for a long time now and I never had any problem with contamination. The cells grew quite fast(2-3 days) and everything was okay.

Since last week I started observing some black dots within the cells. They have a relatively small size but they are not all the same and they are not moving. The media changed color from Friday to Monday from red to yellow, and they are definitely fewer cells that expected. The media even though it is yellow seems clean. The thing is when I first saw these dots since neither me or my supervisor didnt know if it was contamination or some kind of precipitation we started with new cells.

But the problem remains.

I just want to mention that not only we started with new cells but we also changed the incubator and cleaned everything.

I am thinking to start a new plate only with media to see if there is something there. Do you have any other suggestions? Would it be meaningful to keep growing the cells but changing the media every 1-2 days just to see what happens?

Thanks.

You need to provide pictures so people can help you. You could try a plate with only media, but this may not help if it is within the cells.

If it's inside the cells it could be mycoplasma-related. Have you ever tested for this?

Thanks Artemis. No we havent actually checked for mycoplasma. Unofortunately we dont have any camera so I can not provide prictures.

Could it be because of the freezing and the thawing of the cells?

Hi,

got some silmilar problems.  But I used 3T3 instead. Maybe it could help somehow.

I tested different media types and FBS concentration.

A mixture of DMEM and 20%FBS resulted in colonies as you can see in the added picture.

The media turns it color real fast (1day) after passaging the cells look normal and start to form the colonies again. I only occurs with this FBS concentration.

Is this a known problem ? Or is there even a name for it ? Are these colonies or contamination ?

Best regards

It definitely does not look like infection and it may be related to the FBS concentration. There is no need to use more than 10% FBS, even at 10% our 3T3s grow exceptionally quickly.

This is not a contamination. It is in all probability overgrowth of cells on these foci into colonies that are morphologically strongly reminiscent of the spheroid growth form as exhibited by stem cells in serum-free culture. Probably the cells are overgrown, although I can't really say why they show exactly this morphology.

Usually I use "only" 10%FBS for 3T3. But Iam playing around with co cultures. So I supply epithelia cells with this medium as well and they feel pretty comfortable :P

Maybe I should decrease the concentration to 15% and see what happen.

By the way to V Black .... does this look like you problem ?