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

Same problem in my JEG3 colture, months ago. I really didn't understand what kind of weird bacteria they were, but the only thing I did to get rid of them was to trash my cells and start again with a new thawed batch.

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I have been having the same problem in my CHO cultures. Tiny rod-like black things stuck to the flask and on the exterior of my CHO cells. I filtered my DMEM/FBS using 0.22um filter and they still appear. Same story as above - no pH change, no turbidity - just some freaking black stuff dancing around as though mocking me.

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Hi all

I've been isolating and culturing neonatal rat cardiac myocytes for a while now. Every now and then we experience a period when cells, from several subsequent isolations, die. We do not see moving particles, but it looks like the cells just fall apart in thousands of little pieces. The microbiology department tested the media for several contaminants but they couldnt find anything. Two weeks ago it started again and it drives me crazy!! After several perfect isolations cells die again, without any visual sign of a contamination. Again we see that cells suddenly die, preferably after putting them on serum free media overnight. We used fresh solutions, new sterilized media, but for some reason we cannot get rid of it. I attached a picture of dead cells. Has anyone seen this before?

thanks!

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I'm wasting months for the same reason.... in some way every time I start a primary culture I find the same microrganisms...rounded and black. sometimes the contamination is bigger. I think it could come from the operating theater.
The problem is that I cannot obtain clean culture... and that is the start point of my research.
somebody has some advice to help me? :(

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Hi Paw

what kind of cells are you culturing? Have you tested your media by your microbiology department? Try to gas out your culturing room and incubator with formaldehyde, to make sure everything is dead. Good luck with everything

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Hello,

I remember something like that happening to me in HepG2 cell culture and a friend of mine in primary hippocampal culture years ago.... it seems to me that someone ought to start analyzing this contamination... who knows? It can become the subject of really interesting and fundable research

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hey guys,
i have been experiencing a very similar problem as the scientist. it has been 6 months since i got a good harvest of cells for my experiments. I work on trabecular meshwork cells which are very similar to endothelial cells. I culture these cells from tissue explants. the infection or whatever it is is slow growing and as described by the rest do not cause turbidity or other changes in the medium. the cells just die when the serum content is reduced or when i starve them of serum before my experiments. they die on me right on the day of my experiments!!!! i thought it could be mycoplasma and did the DAPI stain but inconclusive. i have prepared fresh medium, and trashed a lot of my dishes frustrating. i am using plasmocin a drug supposed to be effective on mycoplasma but in vain. my cells look totally strssed and have lost their normal morphology. i am not able to track the source of the infection. it could very well be from the outside air cause i harvest these explants under a microscpe in the open.....
my colleagues culture corneal endothelial cells and epithelial cells and they dont seem to face this problem at least they dont have to wait for freeking 3 weeks before they discover that the cells are dead.

this is very frustrating and my research is going no where...
please help!!!

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hi all ! even iam facing the same problem .... no turbidity .... no color change of the media but there is contamination. i can see bacterial cells just as speck, wriggling, multiplying . i gave different antibiotics treatment (steptopenicillin, ampicillin, tetracyclin, streptomycin,kanamycin, gentamycin, norfloxicin) none of these antibiotics are effective........
i checked FBS. same FBS is used to prepare other media. its absolutely fine.
someone told that this contamination results from skin.... may be i got this contamination from mice skin while removing spleen.
does any one have any suggestion to remove this contamination? iam loosing my precious fusion products

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Dear All,

This is a common problem when isolating primary cells. YOU CANNOT WASH AWAY THE CONTAMINATION. YOU CANNOT USE ANTIBIOTICS TO ELIMINATE THE BUGS.
The only 2 things you can do is to CHUCK THE CELLS AWAY and START AGAIN and improve your ASCEPTIC TECHNIQUE.
In our lab we have years of experience going to abattoirs to collect porcine aorta's. Abattoirs are not a clean environment but we can get clean cells. If you cut corners with your technique you will get contamination.


Work in a clean environment ia a sepaerate area of a main lab.
Wash the primary tissue as much as possible before starting the isolation with PBS "A"
Keep everything separate i.e. do not "pool" the cell suspensions for 3-4 days to isolate any possible contamination.
Keep incubators free from contamination, change the water once/week and spray all flask's in and out of the incubator.
Wear a seperate lab coat in tissue culture areas.
Do REGULAR mycoplasma testing i.e. Agar Growth assay test.
Concentrate on isolating the cells and do not try to do other things in between i.e. run a western.
Learn good Ascetic Technique from a hardened professional. I have been in too many labs where you are given a protocol and left to get on with it.

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Hi,
I have cleaned my incubator and the hood many times now. i have prepared and discarded the media over and over again. one thing i dont understand is this.... why does serum starvation aggravate the condition and cause sudden death of the cells. they grow fine until i make them serum free. why does this happen????

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