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kind of contamination?


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#1 prof. moriarty

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Posted 18 March 2010 - 09:06 PM

so i went to the lab to check on my cells (macrophages) yesterday and....hugely obvious contamination in one of my dishes, black stringy stuff everywhere (clearly visible with naked eye) i'm guessing this was fungal contamination.

a second dish had some questionable stuff in it as well....not sure how to describe it, but when i angled the dish to cause the medium to move back and forth some milky stuff was slightly stickng to the dish - again, didn't even need to look through the microscope. threw both of these away immediately. not to mention the absolutely disgusting odor for both of these. terrible stuff.

as for my third flask, there is this huge piece of debris or SOMETHING in the medium. other than that, everything looks fine - aside from slightly cloudy but i think that's pretty normal (i hope - if not, that would mean everything i've been doing for the past couple weeks has been contaminated).

fourth flask looks fine, aside from slightly cloudy as i said before.
i have kept both the third and fourth flasks in the incubator as i am not quite sure they are contaminated. yet.

i sprayed down my hood and incubator with 70% isopropanol. my humidity pan has copper sulfate in it so i doubt contamination is coming from there. i am the only one using the incubator and hood so it's not from other cell lines.

i am a newcomer to cell culture (current student) so i suppose it would come down to just bad aseptic technique.

does anyone have any experience with huge pieces of debris in terms of contamination?
also, is slightly cloudy medium necessarily bad? the medium is an orangey-reddish color. i can see lots of cells floating - the cell line is supposedly semi adherent so that doesn't worry me too much, it's just that huge piece of debris.

additionally, what is the best way to test for mycoplasma? i would do direct culture but i don't have any mycoplasma samples sitting around to compare with...


thank you!

#2 jakatta70

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Posted 19 March 2010 - 12:42 AM

Hi,

I used to work with THP-1 monocytes/macrophages and I found that the media is "milky" when the cells are in suspension and at a high density. The media might turn milky if you had a bacterial infection in the flask but not sure.

I would throw out your media and the cells/flasks etc and take your time decontaminating the hood. Spray everything down with ethanol and a fungicide and it might be a good idea to have a more experienced lab member watch you perform cell culture and then provide a critique of it. Ive been doing cell culture for a few years now but I was in doubt recently about my aseptic technique and asked to be monitored briefly.


Where testing for Mycoplasma is concerned there are several commercially available means to use, each with their own advantages and disadvantages. I have attached a PDF document for you to look at. We use a standard PCR method but this can sometimes give you false positives. Other methods are both time consuming and expensive.

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#3 rhombus

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Posted 19 March 2010 - 03:52 AM

Hi,

I used to work with THP-1 monocytes/macrophages and I found that the media is "milky" when the cells are in suspension and at a high density. The media might turn milky if you had a bacterial infection in the flask but not sure.

I would throw out your media and the cells/flasks etc and take your time decontaminating the hood. Spray everything down with ethanol and a fungicide and it might be a good idea to have a more experienced lab member watch you perform cell culture and then provide a critique of it. Ive been doing cell culture for a few years now but I was in doubt recently about my aseptic technique and asked to be monitored briefly.


Where testing for Mycoplasma is concerned there are several commercially available means to use, each with their own advantages and disadvantages. I have attached a PDF document for you to look at. We use a standard PCR method but this can sometimes give you false positives. Other methods are both time consuming and expensive.



Dear All,

I apologise to other posters who have read my posts before on Mycoplasma detection and testing, but as this post suggests, there are still some unaware reseachers who are compromising their experiments.

PCR is not a valid test for Mycoplasma. Jakatta70 is completely wrong in what he has stated. The problem with PCR is that it gives too many FALSE NEGATIVES. That means people have tested their cells using PCR and think that they are clean, when really they are using contaminated cells. The Food and Drug Administration (FDA) has not licensed PCR as an approved test for Mycoplasma detection for this reason. It also is a very INSENTIVE test i.e. the cells have to be massively contaminated to get a positive.

"Other methods are time consuming and expensive".....this is complete rubbish. We in our Institute use an independent contracting company that charge 80/cell line. They use solid agar method as their detection method. This price includes the courier to pick up the cells from us and deliver it to the company. If the cells are contaminated then we are informed of this within 2 DAYS. The test itself however runs over a 4 week period. So to get a definitive negative takes 4 weeks. The reason why it only takes 2 days to get a positive is that it is a very sensitive test so. This means it will pick up a very low contamination. THIS TEST IS APPROVED BY THE FDA.....so if you are taking a drug or being given a vaccination.... THIS IS THE TEST THEY USE.

Finally, the American Tissue Culture Collection (ATCC)...the largest supplier of commercially available cell lines IN THE WORLD.....so they should know what they are talking about.....NO LONGER OFFER PCR AS A MYCOPLASMA DETECTION METHOD.....because of the large number of false negatives you get with this method. They advise using solid agar in combination with Hoescht staining, again the only other FDA approved method. Hoescht again is less sensitive than solid agar and therefore this method will only pick up a medium to high level of mycoplasma contamination.

I hope this explains current thinking on Mycoplasma testing/detection methods. IT IS ONE OF THE MOST IMPORTANT THINGS TO TEST FOR BEFORE DOING ANY SCIENTIFIC RESEARCH USING CELLS. Gene arrays for example can cost many thousands of pound/Euros/Dollars. 80 to test whether a cell is contaminated is peanuts compared to this. You cannot publish data with contaminated cells in any journal that I know of.

I hope you find this advice useful.......32 years of cell culture experience.......and many co author publications.

Kindest regards.

Uncle Rhombus




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