cells are sick. Please help!!!

I found many black tiny precipitates in my tissue culture plates.  The medium still clear.  The cells seem still growing, but for some cells  the nucleous is not clear and the cell membrane seems like broken already.  No virus was used in the same tissue culture room.  Would you please tell me what the problem is?  Macroplasm contamination?

Many thanks,

SD

Hi SD,

Black specks are not completely uncommon and they may mean nothing. If the media you are using is new (different batch) then it is possible that it is just a particulate from the media. While most media are supposed to be complete free of any solids, it is possible to get some in a given lot.

If these black specks are not affecting your cells and the quantity is small I would not worry about it. Typically it will just go away after a couple of passages (if using different media). Alternatively it may be something your cells are secreting (if these black specks seem to be surrounded by something then it may be that they are vesicles). If that is the case again passing the cells may solve the problem (assuming the cells are not being exposed to some kind of stress, including inadequate CO2 levels). 

Ultimately if the number of specks is not increasing then it is very likely this is not some sort of contamination. Another trick you could do if these specks are mostly "floating" around: change the media a couple of times to wash your cells. This should remove most of the specks.

Hi Ivan,

Thank you very much for your response.  I think stress might be the reason because this happened to other lab's culture as well, and the common thing we used is the CO2 supply.  I will check it out.

I really appreciate your help.

SD

Please  rule out the chances of fungal growth too

I get this from time to time too. What I see is that the dark "precipitate" is very regular - that is, the size of each bit is similar and (more worryingly) often you can see two bits the same size bound together (as if they have just divided!). Then there is the movement: Initially it looked like the particles were moving around, bumping against adherent cells, into other particles etc. Finally, there is the density issue. The things I see are more dense than the cells so if your cells are non-adherent it is difficult to do a low g spin to clean up the culture.  If the cells are adherent, you can just remove the medium, wash once with PBS and add fresh medium.

I did worry about this being some form of contamination which we began to suspect came from primary mouse cell cultures being used in the same TC hoods and incubators. However, I now believe that this is simply cell debris - possibly nuclear as they look a bit like nucleoli. To get this you must also have dead cells - did this happen after thawing a vial of cells? Also many cell types do not "like" being seeded out too sparsely/ They get stressed, some die and you get the "bits".

I did a Google search to see if anyone else had seen the same thing -  and they had. One guy described it as " just some freaking black stuff dancing around as though mocking me.", which I can begin to identify with!

http://www.protocol-online.org/biology-forums/posts/4305more2.html

Thank you very much for your responses, Hilltrekker and Parvoman!

My case is exactly like what Parvoman describled.  The big nucleoli look like being broken into small ones, and the small black dots are moving like they are dividending, so I think the cells were attacked by something.  This phonenone was first observed in the old cultures, then later I saw it in freshly thrawn culture as well.  This also happened to my neighbour lab with which I shared the same tissue culture room, but we used different hoods and incubators.  This started 2 weeks ago.  Probably there is something wrong with the whole room.  I checked the CO2 supply as Ivan mentioned, but it looks fine.  I  grew cancer cells, like MCF7 and H1299, and my neighbour grew dog cell lines as well. 

I will wash the cells intensively with PBS and do several passages and see if the black precipitates will disappear.

THanks again,

SD

If you are that concerned about contamination, simply plate some supernatant from a culture on microbial media and incubate.  That will give you the most definitive answer to your contamination question.

Thanks for your suggestion, D-Fresh.  The media of my culture is clear, so bacteria contamination is not a concern.  Since the cells are still growing normally, macroplasm contamination doesn't seem to be the problem either.  I am treating the cell with all fresh media and washed them intensively with PBS and hope the cells will be happy again.

SD

Hi Sd and Parvoman

I have observed similar black dots in my cultures for both suspensiion and adherent cells. Intiially I got crazy thinking its contamination. I tried to check for bacterial, fungal and even mycoplasma contamination. But nothing was positive( good for me).  As I was handling these cells for first time it added to confusion. Finally I came to conclusion that the spots are nothing but CELL DEBRIS.  It especially occurs when cells are either overgrown or under some sort of stress. After PBS wash and 2-3 passages dots will disappear.

Hi Shubhangi,

It  is a relief to read your message.  My culture is getting better.  After PBS wash and low speed spin (600rpm), there are much less black dots and much more cells with intact nucleoli.  I think  the cells are under a kind of unknown stress.  The CO2 supply is OK and all media is fresh, and today I will inactive the FBS again although the company claimed they have done that already. 

Really appreciate your input.

SD

Unless you are actively working with Abs or the C' system, do not overtreat your serum - remember it contains a bunch of heat labile growth factors too. Check this comparision at Hyclone: http://www.hyclone.com/new_sera/hi/heat_inactivation.htm

We maintain cell lines as well as perform primary cultures of rat as well as human biopsies and this kind of pattern is not common for all the cell types. Rapidly dividing cells show more cellular debris as compared to the slow turnover ones and also the pattern of contamination by bacteria and fungus are altogether different as bacerial cell will make the media more acidic while fungal growth will make it basic. however none of it was comparable to this so called CELLULAR DEBRIS.

washing with PBS or HBSS regularly is a practice we follow as more dead cellular material can elicit growth of contamination too.

Gaganjot Singh

Hi Gaganjot Singh,

Thanks for sharing your experience.   From your message, I learned more about bacteria and fungus contamination now.

SD

Unless you have a specific reason for heat inactivating your FBS, don't do it.  The entire reason you are using (and paying the price) for FBS is becuase it is so rich with growth factors, hormones, etc.  Heat inactivation does just that, inactivates those valuable molecules, rendering your FBS fairly impotent. 

From an ATCC FAQ:

ATCC does not routinely use heat-inactivated serum unless specifically required for a particular cell line. Heat inactivation is usually unnecessary and can be detrimental to the growth of some cells. It will reduce or destroy growth factors present in the serum.

Heat-inactivation (heating to 56°C for 30 min) is done to inactivate complement, a group of proteins present in sera that are part of the immune response. This is sometimes important for cells that will be used to prepare or assay viruses, used in cytotoxicity assays or other systems where complement may have an unwanted influence. Heat has also been used to destroy mycoplasma in serum. Because most serum suppliers filter through 0.1 µm filters to remove mycoplasma before distribution, this is not usually necessary. Heat-inactivation is also recommended for growing embryonic stem cells [p. 75 in Rudnicki, M.A., and McBurney, M.W. (1987) Teratocarcinomas and Embryonic Stem Cells - A Practical Approach (IRL Press Ltd., Oxford)] as well as for many insect cell lines (Weiss, S.A., et al. (1995). Meth. Mol. Biol. 39:65). Heat inactivation will reduce or destroy serum growth factors, and should only be done when there is a compelling reason.

Thanks for the important message, D-Fresh.  I will only do the inactivation for the MEFs cells in the future.

SD