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#1 SciCell

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Posted 02 December 2010 - 11:43 AM

Hello All,

I have 6+ years of cell culture experience but now in my current job I have ALL my cultures tested + for Mycoplasma. So Please I look forward your

1) Suggestions
2) comments how to handle this embarassing situation
3) Precautions
4) How to prevent this situation in future.

might also be fired with this problem because ours is a small start up where everything matters.

Thanks

#2 labrat612

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Posted 02 December 2010 - 01:46 PM

First off,

take a deep breath and exhale.

where did you get your cultures from? And what cell lines are you using? There are some cell lines that are more prone to mycoplasma then others. If you bought it from a company, then there is the possibility that you received it from them. How soon after did your cultures become positive? Soon after you received them?

What test(s) are you using to determine mycoplasma presence? There are some tests that have a higher false-positive then others.

Look at your area- do you have many people running from other areas of the lab through your nice, sterile cell culture area? They maybe your source.

As I'm sure you have already done this, but get rid of your media, commonly used reagents, and cell lines and purchase/ thaw new ones.

It is possible to "clean up" your infected cultures, just be aware that treatment with these potent antibiotics can have other effects on your cells.
I've read in a few places that you can even pre-treat cultures with a substance to prevent mycoplasma contamination. Whether that works or not, I can't say.

Hope that helps!

#3 SciCell

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Posted 02 December 2010 - 02:43 PM

First off,

take a deep breath and exhale.

where did you get your cultures from? And what cell lines are you using? There are some cell lines that are more prone to mycoplasma then others. If you bought it from a company, then there is the possibility that you received it from them. How soon after did your cultures become positive? Soon after you received them?

What test(s) are you using to determine mycoplasma presence? There are some tests that have a higher false-positive then others.

Look at your area- do you have many people running from other areas of the lab through your nice, sterile cell culture area? They maybe your source.

As I'm sure you have already done this, but get rid of your media, commonly used reagents, and cell lines and purchase/ thaw new ones.

It is possible to "clean up" your infected cultures, just be aware that treatment with these potent antibiotics can have other effects on your cells.
I've read in a few places that you can even pre-treat cultures with a substance to prevent mycoplasma contamination. Whether that works or not, I can't say.

Hope that helps!


Thanks for your reply. But I am sure this particular contamination came from 'my' cultures only because I used the same parental cell line to generate other Ab producing cell line, a few months back which came negative and this one NOW is positive. I think our guy uses Mycoalert from Lonza.
Is there any way, I can test the frozen stocks without putting them into culture?

Thanks again!




#4 labrat612

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Posted 06 December 2010 - 08:04 AM

The current techniques for testing the presence of mycoplasma contamination all revolve around using live cultures. I don't believe there is a technique for testing frozen stocks.


Good luck!

#5 rhombus

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Posted 08 December 2010 - 09:09 AM

Hello All,

I have 6+ years of cell culture experience but now in my current job I have ALL my cultures tested + for Mycoplasma. So Please I look forward your

1) Suggestions
2) comments how to handle this embarassing situation
3) Precautions
4) How to prevent this situation in future.

might also be fired with this problem because ours is a small start up where everything matters.

Thanks



Dear Scicell,

1.Please search on this forum for "mycoplasma testing"...there should be alot of post's from me on the subject.


2. At least you have tested the cells...alot of people do not.


3. Quarantine all cells and get them regularly tested by an indepedent and therefore unbiased expert company.


4. Only use the Solid agar test in combination with Hoescht staining.
Only use good quality FBS/FCS that has been properly tested.
Use good aseptic technique and regularly test class II cabinet for product protection.

If you are not the head of the lab, then you are not responsible as all these above procedures should have already been in place.

I hope this is useful.

Kindest regards.

Uncle Rhombus (33 years cell culture experience).

#6 SciCell

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Posted 09 December 2010 - 03:01 PM


Hello All,

I have 6+ years of cell culture experience but now in my current job I have ALL my cultures tested + for Mycoplasma. So Please I look forward your

1) Suggestions
2) comments how to handle this embarassing situation
3) Precautions
4) How to prevent this situation in future.

might also be fired with this problem because ours is a small start up where everything matters.

Thanks



Dear Scicell,

1.Please search on this forum for "mycoplasma testing"...there should be alot of post's from me on the subject.


2. At least you have tested the cells...alot of people do not.


3. Quarantine all cells and get them regularly tested by an indepedent and therefore unbiased expert company.


4. Only use the Solid agar test in combination with Hoescht staining.
Only use good quality FBS/FCS that has been properly tested.
Use good aseptic technique and regularly test class II cabinet for product protection.

If you are not the head of the lab, then you are not responsible as all these above procedures should have already been in place.

I hope this is useful.

Kindest regards.

Uncle Rhombus (33 years cell culture experience).


Hello Uncle Rhombus,

Thank you very much for your reply. I think I did search on this forum too. Anyways, I'll search again. The reason we discovered contamination is that some one from Lonza came in for 'demonstrating mycoplasma kit' and I just submitted a couple of my samples for demo. We found out from checking our records that one of the parental cell line vial which I thawed a few months back is contaminated with Mycoplasma (I don't understand why that was retained in the cell bank stock). I generated the supernatant from this cells and used it for ALL other cells - hence this massive contamination. Well, I am happy for the happy ending - at least. So I am back in business.

To All,

From my experience, I am posting a few words / cautions regarding mycoplasma.

1. Mycoplasma contamination is not easily detectable under microscope (which everyone know) but if the cells are growing SLOWER than usual, we should suspect something and be extra-cautious i.e. not using the media from the same bottle for other cells etc. Though SLOW GROWTH is the key in mycoplasma infected cells, my cells - some were growing normally but some were pretty slow!!
2. Even if you receive the cells / vial from one of your lab members - do at least in-house mycoplasma testing before using it for major experiments.
3. I am not sure but I think the cells look different like having a lot of debris / unwanted material floating in the culture media - no color change (which everyone know).
4. Throw away all the banks of suspected cells.
5. Lastly, the way people treat you is ---- As though you yourself are a mycoplasma agent walking towards them. This takes some months to go away - or maybe will not go away at all.

By the way, the LONZA kit is just great!! -- OK I wrote this too early. Uncle Rhombus has really given good explanation in his earlier posts. All, please do read uncle Rhombus posting by searching this forum like he told. - Regards.

Thanks,
Scicell

Edited by SciCell, 09 December 2010 - 03:16 PM.





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