Contamination issue in cell culture - (Nov/16/2004 )

not too small for yeast. I agree - I think they're yeast

please remember, your skin is loaded with yeast. if you're not perfectly careful about your hands and such, you can contaminate your cultures quite easily with yeast

hey these black dots are cell debries and this is a symptom of microplasma
i had same problem and i confirmed it by DAPI staining that there are some dapi stained particles near the nuclei

Hello everybody,

I have the same problem as most of you. I work with murine lymphocyte B cells (WEHI231) for a year now and I started having this problem, when I moved to another lab at our Faculty. There is a different microscope that enables you to see the background around the cells much clearer than the microscope I used to work with. I started to see in my culture very tiny spots (black or very bright-it depends of the microscope settings), that vibrate or move (you can see that at 400x magnification). At first I taught it was cell debris, but after 10-14 days it would not go away and it seemed to multiply. I have now unfrozen at least five new batches, remade fresh media and started all again, but it wont work. I moved again to the old lab, which is much cleaner, but the problem wont go away. It is also possible, that I had this problem before, but I just didnt see it.
I will try to test my FBS as some suggested, clean the lab and start again. I am really tired of this, because I have been cleaning and throwing away stuff for the last three months!

I am attaching two pictures, taken one after another to show the movement of the "particles". They appear as dots, rods or a chain of two or more dots.
The cells are qrowing very nicely, they do not seem affected by the "particles". The medium is clear, there is no change in pH (but this may be due to the small number of particles - because they seem to grow very slowly).




bRuLa

Hello, me again
I did nuclei staining with Hoechst and watched the cells in live-view. I was really surprised, because everything moves and vibrates A LOT, including the cells (I have suspension cells)! The tiny black dots move as hell, but they do not stain with Hoechst! Actually, I saw black "shadows" of the dots over/near the cell's nuclei (you can see this in the 2nd picture, bottom left). This means it is just cell debris. I suggest everybody to stain with DAPI or Hoechst before making conclusions. I made a lot of panic in our lab, because everybody sdtarted seeing the moving dots! Moreover, when you watch at higher magnification (1000x) you can see the dots are actually not so perfect/roundly shaped as they seem at 400x.
I include some pictures (I added 2x concentration of Hoechst as usually do and the exposure time was very high too, so I could not miss the spots if they would appear).

bRuLa

[attachment=3957:wehi231_...okuzbe_c.JPG]
[attachment=3958:wehi231_...okuzbe_d.JPG]

[attachment=4215:A___21_f..._535_50_.jpg]Hi,

I'm new on here and the problems mentioned here grabbed my attention. Recently have a new osteosarcoma cell line that overexpresses a GFP tagged version of my protein of interest.

Since thawing out the vial sent from the company I have noticed these so called small black dots/bits floating around that people here have been mentioning. However I assumed they were cell debri but am now not so sure. I can see them at x10 magnification (so they must be pretty big right?!) and only seem to move when I disturb the flask. cells seem fine (no changes in growth, morphology or response to known treatments), no changes in ph media, no turbidity etc just as what has been mentioned on this topic here.

However when I image them to investigate localisation of my GFP tagged protein (stably expressing...I do not transfect in this construct and no staining with primary/secondary antibodies involved) I see green dots everywhere both in cell and outside (see attached picture) which made me think the samples were contaminated (especially in those in antibiotic free media but not really in Pen Strep containing media) BUT I can't pick up anything other than the nuclei in the DAPI channel and I thought if contaminated there was a good chance of it coming up after DAPI staining. So very confused and concerned.

I have been doing a lot of cell culture for about 2 and a half years now including in a lab that never used antibiotics as standard and never have had a problem. Whilst I am still new to this I am very careful with sterile technique- I have watched my colleagues and have been observed by more senior staff doing tissue culture and they cannot see anything I appear to be doing wrong. I follow all advice and things I have learnt from people and am very careful but am now worried that my technique is still not good enough but have no idea what I may be doing wrong.

Any ideas on this and the possible contamination please?

Cheers

I never noticed this "contamination" either until recently. I acquired some MEF cells and there were so many small black things floating around. My PI had said bacteria should move/vibrate really fast, and these black things appear to be doing that. But, I checked with the company and these black things were just cell debris. I have seen a bacterial contamination and they move much more quickly than these small black things. The black stuff just move in a circle in place, but the bacteria are worse and have directional movement (from what I recall), and there would be lots and lots of them, and the media will begin to look a bit cloudy. I attach an image where I stained with lectin, and you can see that in the background there is some bright green dots showing up. @Mojito - I think your image was taken at a high exposure? There seems to be lots of background, so I assume there are not as many dots if you take an image normally, right?

Do you all use water bath?...then don't ......water bath is a huge source of contamination. even if you use tablets and then spray 70% alcohol the bugs are still there.

In England (Essex University) we had a special incubator (or better call it a warmer) instead of water bath and we never experienced contamination.

These little bastards made me quit a research on Glioma Primary cell culture as they just didn't die. I even filled up the whole flask with only Antibiotic cocktail and after that added 70% alcohol but after 30 min they were still moving.

I assumed they are cocci......we also had contamination with Baccili.....however baccili move faster, you can easily see them they run between cells...but cocci just sort of vibrate in their place.

trust me, they don't die.

Hi, I'm another victim of this weird contamination, I don't work with primary cultures though .l Mine are MA-104 clone cells infected with the parasite Neospora, and since we obtain the isolate from mice to further adapt them to in vitro culture I guess some sort of contamination is coming out of this animal model. At the beginning we didn't figure we had this contamination because we usually work with medium containing Pen-Strept-Fungizone, but now we try to keep cultures without antibiotics and we have started to see at this tiny "bugs" with irregular shape, either alone or glued each other and sometimes adhered to the cells. At first, cultures look fine and medium has the appropriate pH but suddenly they raise their number and pH drops to 5.5 (amazing yellowish medium), so I must get rid of flasks before contamination affects the rest of what we keep on the incubator. We have run several tests (agar and brot culture, PCR for mycoplasma detection) and nothing shows up. If someone looks at the video and photo we are attaching, I would appreciate any advice you can give. Thanks!!!

Hello All,

I am having mold problems with my primary cell culture. The first time we got struck with mold, I got rid of all the cell flasks, thoroughly cleaned the hood and autoclaved everything in it. I also cleaned out the incubator, autoclaved shelves etc, wiped down all walls and doors with 10% bleach, sterile DI, and then 70% ethanol. I also changed the HEPA filter and the air filter stop cork. I also have been extremely conscious of my asceptic technique...all to no avail!
After cleaning out the lab, I seeded some PANC-1 cells and MCF-7 using my hood and stored the flask in an incubator in a lab on a different floor. Everything seemed to be going wonderfully. I split the PANC-1cells and no signs of problems. I then introduced three more cell lines. Still no problems. I then split the PANC-1 cells again (passage 3, all other cells lines still on passage 1). I then shifted a couple of the passage 3 PANC-1 cells and the flask of MCF-7 cells into my now immaculately clean incubator and left one PANC-1 flask back in the incubator in the other lab with the remaining cell lines. Everything seemed to be going ok for the 1st two days. On day 3, I noticed mold in only one of the panc-1 flasks in the incubator in my lab. More interestingly, there was mold in the only panc-1 flask in the incubator from the other lab. Note that all this while, media changes are being conducted in the same hood.

I put some panc-1 media in my incubator overnight - no problems.

So I am not sure what to do next about getting rid of this pesky mold. I doubt it's my hood since the other cell lines have not been affected as yet. However, the other cell lines have also not been split as yet. So I am guessing that the problem might be with my splitting technique? I was going through the messages here and I am guilty of putting my pipettor inside my culture flask while transferring trypsinized cells into new culture flasks. Could this be a potential contamination source?


Any other suggestions on how to narrow in on the cause of contamination?

I have attached some pictures as well.

Can someone help identify what contaminant this is in my cell culture? See attached.

It usu. occurs as diploid dots, much smaller than cells (eg. 1um), usually moves in circular fashion, and grows exponentially fast especially in Fetal Calf Serum. I filtered my media, thawed new cells, used PenStrep antibiotics, but I still see these contaminants grow alongside my cells. My Jurkats no longer clump as they'd used to and and replication is almost non-existent.

If someone can identify them, please help! Thanks!

-Jason