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Cell Culture Contaminationlcoyne2004-08-26Click here to register.
Hi, I've been having major problems growing cultured cells for the last 6 months. The cells seem to be contaminated with a microbe that passes through a 0.1 micron filter. I have even observed these microbes moving about inside of the cells. I have tried incubating each media component alone, and have found that fbs from three separate sources all have these microbes. Has anyone else had these sort of problems, and if so, how did you solve it?
 
Re: Cell Culture Contamination
yasser2004-08-30Click here to register.
It seems that you have a problem due to the FBS
you can prepare the medium and filter all of the media through Stericup,
try to use a suitable Antibiotic witth the medium like Penicillin/streptomysin in the optimal cocentration .
If you are using Trypsin to shear your cell Filter it by 0.2u filter ( whole of the stock) before doing splitting into small aliquites and freez them down.
and hopefully this can work
if not try to know hwat is that microbe because may be it is Mycoplasma and you need to treat the cell line it self
 
Re: Cell Culture Contamination
lcoyne2004-08-30Click here to register.
Thanks, I'll try that.
 
Re: Cell Culture Contamination
ryanj2004-09-16Click here to register.
I think you may not have a true biological contaminant, The chances of a microbe other than a virus passing through 0.2 micron filters is very small. i would run a dna staining test on the cells to see if these particles in the cells actually have DNA. There is a protocol on the Corning Technical web site www.corning.com/life sciences.

John
 
Re: Cell Culture Contamination
Yongabika2004-09-20Click here to register.
Hi all,

Perhaps,it is not too late to make anote now. There is
a need to try to identify the contaminant,this is
crucial for future work. My suspicion is it could be a
mycoplasma as suggested by John and others. But what if
it is a rickettsia or chlamydia?

In any case,I would suggest a bacteria culture test on
a broad range of media,in addition to a DNA analysis
as suggested by John.

Regards,
Kenneth Yongabi
Abubakar Tafawa balewa university,
ZERI Research Centre,Bauchi,Nigeria
 
Re: Cell Culture Contamination
lcoyne2004-09-20Click here to register.
I've tried treating these cells with plasmocin, and these small 'black dots' do not decrease in number, so I don't think I have a problem with mycoplasma. I have had limited success treating with tetracycline - they are reduced in number, but not completly eliminated, which suggests some sort of microbe. They can also be seen 'wriggling', both in the media alone and inside of some cells. The microbes are able to grow seemingly only in the presence of FBS.

Has anyone ever came across an organism like this?
 
Re: Cell Culture Contamination
Yongabika2004-09-21Click here to register.
Hello lcoyne:

The FBS media is rich and thus support the growth of
the microbe which I think it is very fastidious.It may
require a stronger broad spectrum antibiotic-Could you
use a quonolone antibiotic like ciprofloxacine? or in
the alternative,add metronidazole(Flagyl)to the
tetracycline ,and let us know your experience.
Whatever the case I do not expect any new bug,rather a
mutant/some strain of an obvious bug.You must run a
DNA and a culture test on a bacteriological and as
well as mycological agars.

I will be glad to get involve in this challenge

kenneth yongabi
medical microbiologist,
ZERI research centre,ATB University,Bauchi.Nigeria
 
Re: Cell Culture Contamination
lcoyne2004-09-21Click here to register.
Thanks for the reply. I will try ciprofloxacine.

I have tried a Dappy stain and nothing appeared. I'm not sure what this means as they do wriggle about.

Leanne
 
Re: Cell Culture Contamination
Yongabika2004-09-23Click here to register.
Hello lcoyne;

That is good, let us see how it goes; I am positive.
You say the organism wriggles about, could you describe
to me the motility? Could you try an electron
microscopy-anyway both light microscopy as well.
Let us have some description of the agent.

kenneth yongabi
 
Re: Cell Culture Contamination
lcoyne2004-09-23Click here to register.
The way the organisms move makes them look like fleas. They seem to 'buzz'. They all seem to move in different directions.
Using an electron microscope, I have now found these things inside of a cell. They are about 1 micron, and spherical.
I have found some literature describing an organism with many of the characteristics I have seen with these bugs - nanobacterium sanguinium.
 
Re: Cell Culture Contamination
Yongabika2004-09-25Click here to register.
lcoyne:

That is great,you may have to keep the isolate,and try
a gram stain.Let us see the response to
ciprofloxacillin.

Kenneth

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