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

Hi

I really do not know if this is the right forum to ask this question. Our lab does a lot of primary cell culture work like T cells and DC. The last week there has been a problem of contamination.
There are wriggly structures, highly motile, no visible flagella, like tiny specks. It was first thought to be cell debris, but they are motile and seem to have penetrated some of the cells or look like swarming inside the cells. Cells look healthy and no pH change in media or obvious turbidity. I have been reading about mycoplasma contamination, however most references state that mycoplasma contamination is usually invisible and detected by PCR kits.
How slowly does yeast grow on primary cell cultures, as some of the cultures were seven day old and it did not look like the organism had taken over even after 24 hours.
could you please help.

A researcher

Are the speckles dark, like small black speckles?
They could possibly be confused for debris, but are actually a
strange strain of bacteria as evideniced by the propigation of the speckles?
It has been my experience that this is not a form of myoplasm, but exhibit
characteristics similar to this. We have found that this form of contamination
comes from a water bath and grows in serum. BAsically, someone contaminated
their serum with water bath water.
If you have these in your cells, you may try to get rid of them by culturing in high doses of antibiotics.
However, we have found that once these little guys are in a group of cells, well basically, resitance is futile. The cells should be trashed. If even a few of these cells are added to a new flask, the bacterium will be carried over on the cell wall (??).

I believe it is kind of contamination coming along with the primary cell isolation. We have isolated BAEC and HUVEC and sometimes same contamination appeared. I do not know the specific name for those organisms as english is not my native language.
What I suggest is to throw the cells away, usually the whole batch because they come from the isolation process. But, if you really want to try to save the cells, try the followings. First, wash cells with PBS extensively. Then trypsinize the cells and centrifuge. Use lowest speed (50g, 2 min) to make sure only the cells were spinned down. Pass the cells in to clean dishes using medium containing P/S, gentamycin, and tetracycline. Fungazon is another choice. Repeat one day after if necessary. After two or three passages, cells will look better, then go back to normal culture. This is under the assumption that your primary cells can tolerate such procedures.
good luck.

Hello,
I was just checking (almost all the net!!) about different existing cell culture contaminants.
Because I observe the same kind of problems in my cell cultures, little spots in the supernatant, where i first thougt about debries and now in the cytoplasma where they seem to multiply... really strange : no pH variation, and really slow growing rateof this spots, so the high doubling rate cells are ok , whereas others slow growing cells are more affected. Mycoplasma test is negativ. And i was looking on my flask during 10 minutes: are they moving or is it just brownian movement...?!!!

I need some help

these motile blacky rod will be gaining their doubling populations in a short time. it was recognized as special gram bacteria (further information: i ;m running some procedures to get them understood, as these bacteria might be appeared during isolation processes, or might be from the cell u r goin to culture( donor variability), under microscope they r moving just like rushing ants, might exert some morphologies adapted in ur medium)....long story, cos the atmosphere especially the nitrogen might be reacted with the cells' during isolation(rarely occur) but in some cases it causes that happen..!

so, keep the cell will definitely waste ur time,ur money as well. i suggest put them all in trash, start the new one. or further ur project on the bacteria as extra parameter!

From: Prof.Sycay

I have been experiencing the same problem. These tiny black speckles (or whatever you want to call them) are motile but no apparent flagella. They cause no pH change nor turbidity in the medium. They have been present on all of the HUVEC lots I've been working with. I'm running a bioreactor with HUVECs and a lot of them are present during cell count (some days are more). They don't seem to affect the cells drastically. But if I could find a way to get rid of them, it would make my experiments smoother. I always make sure that the materials I use are sterile at all times. I don't know how to illiminate them from my cells. If anyone has the answer please share it with us!!!

I am also familiar with this particles. I couldnt find any logic that will explain these particles. They seem to appear regardless of cell batches or medium. sometimes their number increases with time and sometimes not.
In order to deal with it i do next things: 1) adding once a week an Aquasan® tablet to water bath on a regular basis and 2) immideately after particles have appeared the incubator is being cleaned in addition to general maintanence of the tissue culture unit.

Maybe I've experienced some kind of this contamination too in a CHO culture.
I noticed the tiny moving specks too, but they seemed to be also inside or on the surface of the cells.
I observed them during some days, keeping on subculturing. There was no medium turbidity, but cells began to look "sick": the extremities strated to be thiner and thiner and little dark spots or grains appeared on the cells surfaces. In the meantime, the specks started to form colony-like groups (like little chains).

Hi there,

I too join the community. same funny organisms. and they sometimes go retard the growth of my cells. and they also sit on the cell surface... i dont see them inside the cells... whatever they are, I just hate them.

- seeker

I have these too! My cells look fine when I first thaw them (they're mouse embryonic fibros) but after a few passages there is an accumulation of debris, little black/phase bright specks maybe a um in diameter all over the cells, sometimes in little clusters, and the cells themselves get long stringy extensions and just look . . . unhappy. I'm studied spreading and motility so needless to say, this is a problem.

Does this sound like mycoplasma? Other contamination? Or just unhappy cells?