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What to do about mycoplasma?


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#1 asta6801

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Posted 13 January 2013 - 04:08 PM

Hi

I've recently started my PhD in a lab where there is a massive problem with mycoplasma. The majority of cell lines in the lab that have been tested have been shown to be positive, including some of mine that I imported from overseas (although I think that they may have been contaminated before I even got them Posted Image ).
The head of my lab just doesn't want to hear about it- it has been brought up several times with him and he dismisses it (he is a bit of a tyrant). He does not want us to throw out contaminated cells, and says that even testing for mycoplasma is a waste of time. I know this is wrong, and my results have been affected by the mycoplama infection in the lines that I'm using.
I guess what I'm asking is this: if I bring up new, uncontaminated stocks of cells, what are the chances that they will catch mycoplama from being in an incubator/hood with other infected cell lines?
And furthermore, is there any way, or has anyone had any success, in curing cells of a mycoplama infection? I have read some reports of it being possible, and have attached a protocol here that I was considering trying as throwing things out is apparently not an option in this lab.

Thanks,

Very worried PhD candidate

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#2 Tabaluga

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Posted 13 January 2013 - 06:18 PM

I use BM Cyclin in my cells too when they are mycoplasma positive. For me, it works. However I cannot say if it's a long-term cure or not, you should test regularly and don't hesitate to start another round of treatment when they become positive again, that's how I do it.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 leelee

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Posted 14 January 2013 - 12:03 AM

Honestly, the ONLY reasonable option is to throw contaminated the cells out. The behaviour of your cells is likely irretrievably damaged, so even if you cure them- you can't trust your results. Basically, results generated using contaminated cell lines are useless.

And no journal will (or they shouldn't) accept papers containing data from mycoplasma positive lines. So either your lab head is intending to lie (major red flag), or wants you not to test so he doesn't have to deal with it (another major red flag).

Is your lab head also your supervisor? If not, I would take this to your supervisor and see what he/she says.

#4 Tabaluga

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Posted 14 January 2013 - 01:38 AM

Honestly, the ONLY reasonable option is to throw contaminated the cells out. The behaviour of your cells is likely irretrievably damaged, so even if you cure them- you can't trust your results. Basically, results generated using contaminated cell lines are useless.

And no journal will (or they shouldn't) accept papers containing data from mycoplasma positive lines. So either your lab head is intending to lie (major red flag), or wants you not to test so he doesn't have to deal with it (another major red flag).

Is your lab head also your supervisor? If not, I would take this to your supervisor and see what he/she says.


Hm, just interested, leelee: what would you do with primary cells that are already contaminated from the very source (patient) ? because that's what I'm working with (not established cell lines), maybe I should have mentioned it in the above post.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#5 asta6801

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Posted 14 January 2013 - 03:26 PM

Unfortunately he is my primary supervisor, but my co-supervisor is aware of the problem and recognizes that it is a big deal. I guess we are just going to have to try talk my primary supervisor around. He has been publishing in journals with ok impact factors like PNAS, don't know how he's been getting around it. Until then, I guess I'll have a go with the BM cyclin and see what happens. Thanks for the help!

#6 science noob

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Posted 14 January 2013 - 03:54 PM

Just curious, what primary cell lines are you working with? Could be due to it's source and non-sterile cell extraction process right at the start.

#7 Tabaluga

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Posted 14 January 2013 - 04:09 PM

Just curious, what primary cell lines are you working with? Could be due to it's source and non-sterile cell extraction process right at the start.


They're brain tumor cells.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#8 leelee

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Posted 14 January 2013 - 07:48 PM


Honestly, the ONLY reasonable option is to throw contaminated the cells out. The behaviour of your cells is likely irretrievably damaged, so even if you cure them- you can't trust your results. Basically, results generated using contaminated cell lines are useless.

And no journal will (or they shouldn't) accept papers containing data from mycoplasma positive lines. So either your lab head is intending to lie (major red flag), or wants you not to test so he doesn't have to deal with it (another major red flag).

Is your lab head also your supervisor? If not, I would take this to your supervisor and see what he/she says.


Hm, just interested, leelee: what would you do with primary cells that are already contaminated from the very source (patient) ? because that's what I'm working with (not established cell lines), maybe I should have mentioned it in the above post.


I would say that these have become contaminated during the isolation process or during culture in the lab (I wouldn't have thought you'd find mycoplasmas in the brain of people without a detectable infection and symptoms, and surely it is quite rare?), so I would work to improve the aseptic technique of whomever is doing the isolation in the first place.

The majority of mycoplasma contaminations come from either contaminated cell lines in the lab- or from lab personnel. In your case, perhaps from the scalp of the patient, if the tumours are not removed with sufficient sterile procedures?

For results generated with the contaminated lines, honestly personally I wouldn't feel comfortable to use them. Or at the very least, I would regard them with a great deal of suspicion and endeavour to obtain more data from uncompromised samples.

#9 Tabaluga

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Posted 15 January 2013 - 04:35 AM

I would say that these have become contaminated during the isolation process or during culture in the lab (I wouldn't have thought you'd find mycoplasmas in the brain of people without a detectable infection and symptoms, and surely it is quite rare?), so I would work to improve the aseptic technique of whomever is doing the isolation in the first place.

The majority of mycoplasma contaminations come from either contaminated cell lines in the lab- or from lab personnel. In your case, perhaps from the scalp of the patient, if the tumours are not removed with sufficient sterile procedures?

For results generated with the contaminated lines, honestly personally I wouldn't feel comfortable to use them. Or at the very least, I would regard them with a great deal of suspicion and endeavour to obtain more data from uncompromised samples.


OK. Unfortunately we have no influence over the cell isolation process (we get them "third hand"), but I'll keep it in mind.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#10 leelee

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Posted 15 January 2013 - 05:18 AM

That's the tough think, isn't it Tabaluga. On the one hand, obviously contaminated cells aren't ideal, but on the other- they're the only thing you've got.

What I would do is do my utmost not to spread the myco from one culture to another, so no shared media, decontaminate the incubator/water bath/hood often and let everyone else in the lab know they need to be hyper vigilant with their aseptic technique too. I've read myco can survive in a laminar flow hood for as long as 6 days. 6 DAYS!! Scary!


Then hopefully if you do get a negative batch they can stay that way. I have my fingers crossed for you. And you too, asta6801




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