You would not be able to see mycoplasma by a 40X objective, nor does mycoplasma contamination generally result in massive cell death. It sounds like you are describing cell debris from an unhealthy culture. A mycoplasma contamination would alter growth rates, cause the activation/repression of genes, among other effects, but it usually doesnt result in total culture annihilation, though I've not worked with mES cells.

While it is a good idea to eliminate the possibility of mycoplasma contamination (and test regularly in your TC lab), it's possible that the conditions for growth of your mES cells needs to be optimized (new media, missing an essential supplement/growth factor, bad batch of FBS, etc).

You would not be able to see mycoplasma by a 40X objective, nor does mycoplasma contamination generally result in massive cell death. It sounds like you are describing cell debris from an unhealthy culture. A mycoplasma contamination would alter growth rates, cause the activation/repression of genes, among other effects, but it usually doesnt result in total culture annihilation, though I've not worked with mES cells.

While it is a good idea to eliminate the possibility of mycoplasma contamination (and test regularly in your TC lab), it's possible that the conditions for growth of your mES cells needs to be optimized (new media, missing an essential supplement/growth factor, bad batch of FBS, etc).

Hi Andrew,

I second your question. We are seeing the very same thing in ordinary 293T cells.

The "spinning" is particularly disturbing...! Although our cells are not dying, quite the contrary they seem unimpressed. No changes in growth rate or attachment. Still, these jiggling/spinning dots are clearly not a normal thing to observe. We use the cells for lentiviral production. Since we've seen the dots, our viral pellets have been larger than normal, and difficult to resuspend. So we think these two changes are connected. We too have been unable to eradicate the dots. They also appear in cultures thawed from older frozen stocks.

Anybody out there who knows what they are?

Hey!

I think this is a very common kind of contamination that is in most cases likely overseen or recognized as debris. I've seen this in my cultures also from time to time - in some cases, if the cells were very precious I tried to get rid of this contamination by extensive rinsing with PBS - sometimes worked and the cell line was clean afterwards.

You may check through this forum, who discussed that issue extensively:

http://www.protocol-online.org/biology-forums/posts/4305more1.html

I copy/paste also a very informative post from this forum, which I believe gives a reasonable explanation:

" I have been experiencing similar problems since the last couple of weeks. I am working with MCF-7 cell lines that I had got from another lab three months back. Evreything was fine till around two weeks back when I started seeing tiny black specs between cell colonies. No pH changes, no turbidity of media. The specs stated increasing in number- strongly adherent, slow growing. I got help from our Department Microbiologist and plated the contaminated cells and media on agar plates and tryptic Soy broth. Sure enough they showed colonies on blood agar, chocolate agar and Tryptic Soy agar after 3-4 days with very strong repulsive smell from these plates. The broths look clear. The microbiologist believes that they are Acinetobacter species- slow growing,Gram negetive, multi drug resistant & could be found in water, soil and is the only Gram negetive bacteria found as a part of normal skin flora!!

I tried Penicillin Strep treatment but they seem to be resistant. Now they have spread to another cell line that I am using- Embryonic Rat cardiomyocytes that I had recently obtained from ATCC.

I have noticed most of them remain adherent to the flask even after trypsinising the cell cuture. I have no clue what to do to eliminate them from my lab before I start a new batch.

Please advice.

drmvk2000"

Best wishes,

Andreas

Hi Andreas,

thank you for this link!

Although I believe these might still be two different things. Our "specks" are not attached to the substrate between colonies, rather they are always associated with cells. They are sort of "dancing" or "spinning" on the sides, or on top of, or perhaps actually inside, individual cells. This rapid, but spatially restricted mobility is their most notable property.

Not all cells are affected. In low-density cultures, we have to look out for cells with specks. In high-density cultures, the specks become more pronounced. We never see them alone, without cells. Supernatants remain clear, no color changes, and no growth of anything upon prolonged incubation of supernatants from "specked" cultures.

We always filter all media including supplements through 0.2 µm, before we use them with cells - basically to remove precipitated bovine serum protein from FCS. We also use the same FCS for other, primary cultures, and never see any dancing specks there. So, we believe that FCS is not the source.

We will do a DAPI stain, to see whether the specks actually contain DNA. I will post the result here.

Thanks again,

Alex

It certainly appears that there are a number of contaminates that fall into this black speck realm, a colleague is searching and reading what they can to get a handle on what might be going on.

Certain we see nothing with media alone, the MEFs that the mES are on are of course aging / dying and so their debris around the ES cells is not helping, it seems yes it is unlikely we are visualising mycoplasma under x40 obj phase contrast (they are apparently anywhere between .3 and .8 micron), we have not looked with DNA staining as the cells were in large flasks, and with that said we are unlikely to track down what this actually is via microbiological examination because of pragmatic issues of the time and the expense.

Like Alex if we work out what we are dealing with I will update here. Though any other inputs / suggestions would be extremely welcome.

PS my colleague is trying to sort out a video of the cells via youtube or some such but is having difficulty with file format and size.

Discription we have here is certaily of mycoplasma. There is no point in trying to make your culture contamination free by antibiotics. Because there is no way one can get rid of mycoplasma, although products are available but in practice they dont work and adds to your problem, and is a waste of time and resources. You may test your culture for contamination if you have in house facility for testing but most contaminants me remain undetectible at earlier stages. Such contaminations are difficult to elimnate, as they could be antibiotic resisitant.

I would suggest you to dicard your culture and media, as it may contaminate other things in your lab, and start afresh using clean culture after following decontamination procedures, as prevention is the best strategy.Testing wil help you in identifying and decontaminating your lab. Good luck!!!

You may go to the fololwing links for detailed information:

http://www.the-iom.org/assets/files/IRPCM_Team_Mycoplasma_contamination.pdf

http://www.dsmz.de/fileadmin/Bereiche/HumanandAnimalCellLines/WebMycoCellCulture.pdf

http://www.roche-applied-science.com/PROD_INF/BIOCHEMI/No.1_96/p48-51.pdf

http://www.atcc.org/CulturesandProducts/CellBiology/KitsPanels/UniversalMycoplasmaDetectionKit/tabid/1789/Default.aspx

Hi guys. I used to suffer a lot from mycoplasma contamination. Once the starting MEF cells get contaminated, you can hardly get iPS cells. The jiggling/spinning black dots, I guess, are not mycoplasma itself, but rather like some kind of secretary motabolites produced by cells under myco-stress. According to my experience, cell cultures with these dots should be dumped ASAP, otherwise other cell cultures could get contaminated as well. These myco-contaminated cells grow slowly and easily get to death, especially upon virus infection or plasmid transfection. Invivogen supplys an agent called Plasmycin which can kill mycoplasm but clearly needs a long time (like 2 wks) and frequently doesn't work well . So my advice is discard them ASAP, clean your hood and incubator and thaw a vial of cryopreserved line. Good luck to you!