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Mycoplasma cleanup time... - (Mar/28/2011 )

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SO, some of our cell lines have mycoplasma. I have narrowed it down to being the hood or the incubator because another girl has used the same tc lab and has her own separate hood and incubator and all her cell lines have been tested negative.

We have been fighting with Lonza as we have some problems with the accuracy of their MycoAlert test which is why we are having so many problems. So currently we are doing mycoalert, pcr and Hoeshct staining for all our cultures.

I don't think it's the incubator as we did a deep clean and autoclaved it (95C for 2 hours, it's a new hera-cell) and it's brand new (well, 3 months old).

The hood had a problem with it and the engineer has just fixed it but previously it was running at a lower exhaust setting. it's a class II microbiological hood and it's only been commissioned 3 months ago.

Would it be worth getting the company out to do a full hood decontamination? We did wipe down all the surfaces with Virkon and 70% EtOH. We don't have a UV light but we have been pretty paranoid.

Any more suggestions anyone? Would it be possible for mycosplasma to infect other cells in the same incubator? Can they jump from one flask to the next? We used plug seal flasks from corning. I'm pretty certain it's the hood....

-PostDocTrauma-

What about your liquid nitrogen tank? I have heard (unconfirmed) that it is possible for mycoplasma to spread via leakage of the nitrogen into the tubes when frozen (that's if you don't store them in the vapour phase).

-bob1-

These cells were frozen down and kept in -80C to avoid the liquid nitrogen issue for now.

It's really traumatic as this is my second postdoc and the first time I'm having such a major issue with mycoplasma :(

-PostDocTrauma-

PostDocTrauma on Mon Mar 28 15:20:05 2011 said:


SO, some of our cell lines have mycoplasma. I have narrowed it down to being the hood or the incubator because another girl has used the same tc lab and has her own separate hood and incubator and all her cell lines have been tested negative.

We have been fighting with Lonza as we have some problems with the accuracy of their MycoAlert test which is why we are having so many problems. So currently we are doing mycoalert, pcr and Hoeshct staining for all our cultures.

I don't think it's the incubator as we did a deep clean and autoclaved it (95C for 2 hours, it's a new hera-cell) and it's brand new (well, 3 months old).

The hood had a problem with it and the engineer has just fixed it but previously it was running at a lower exhaust setting. it's a class II microbiological hood and it's only been commissioned 3 months ago.

Would it be worth getting the company out to do a full hood decontamination? We did wipe down all the surfaces with Virkon and 70% EtOH. We don't have a UV light but we have been pretty paranoid.




Any more suggestions anyone? Would it be possible for mycosplasma to infect other cells in the same incubator? Can they jump from one flask to the next? We used plug seal flasks from corning. I'm pretty certain it's the hood....


Dear PostDocTrauma,

Well were do I start:

The only valid tests are Hoescht staining and Solid agar. These tests have to be done regularly. Mycoalert is not a FDA approved test.

Always use an independent lab to do the testing....no vested interests involved.

Any cells that are not tested have to quarantined and then tested.

Any cells found to be positive should be CHUCKED AWAY....the damage has already been done.

We have 30 years experience in this field..... contamination has not (in 30 years) "jumped from one culture to another". We have had cells that are positive, grown in the same class II cabinet/CO2 Incubator, and never seen cross contamination.

I have looked after 4 liquid nitrogen dewars for 30 years....in liquid phase....again there has never been a cross contamination.

You cannot "Autoclave a CO2 Incubator".....it is a high temperature cycle that does not decontaminate the interior, but does help keep any possible contamination at low levels.

I have not used UV in 34 years in the lab.....in fact the first cell culture I did was on the bench with a bunsen burner. UV IS NOT REQUIRED it just breeds bad practice.


I would be very happy to answer any further questions via a private message if required.

Kindest regards.

Uncle Rhombus.

-rhombus-

thanks rhombus. :)

We were just eliminating every possibility. We can't afford to send it out to another lab for testing. we are a completely new lab and have received 44 cell lines from different collaborators. (that's £44 * 85/test = £3740 just for testing one time....) (also, just wondering why a test has to be FDA approved? It's just for research right? MycoAlert is sensitive and tests a large number of cell lines, followed by a Hoescht stain it's pretty foolproof?)

we don't have a quarantine lab, we just have one tissue culture room with 2 hoods and 2 incubators so we try and keep everything seperate until they have passed the mycoplasma test.

I have been looking through and wondering where they are coming from but with cells from 3 different collaborators being thawed this time and all 5 cell lines being positive, it does look very much like we are the cause of it, SOMEWHERE in our lab. the common denominator is that the ones thawed were frozen down in our lab before the big clean.

So i'm grasping at straws and running our of ideas <_<

-PostDocTrauma-

PostDocTrauma on Tue Mar 29 15:02:48 2011 said:


thanks rhombus. :)

We were just eliminating every possibility. We can't afford to send it out to another lab for testing. we are a completely new lab and have received 44 cell lines from different collaborators. (that's 44 * 85/test = 3740 just for testing one time....) (also, just wondering why a test has to be FDA approved? It's just for research right? MycoAlert is sensitive and tests a large number of cell lines, followed by a Hoescht stain it's pretty foolproof?)

we don't have a quarantine lab, we just have one tissue culture room with 2 hoods and 2 incubators so we try and keep everything seperate until they have passed the mycoplasma test.

I have been looking through and wondering where they are coming from but with cells from 3 different collaborators being thawed this time and all 5 cell lines being positive, it does look very much like we are the cause of it, SOMEWHERE in our lab. the common denominator is that the ones thawed were frozen down in our lab before the big clean.

So i'm grasping at straws and running our of ideas <_<


Food and Drug Administration (FDA): All drugs/vaccines have to be tested by FDA approved mycoplasma tests. They are the most sensitive and reliable. This is why we use them in our research institutes. This is why the American Tissue Culture Collection (ATCC) use them exclusively for their testing....remember that the ATCC are the worlds largest supplier of cells. The ATCC no longer use PCR testing for the reason that PCR gives too many false negatives......the same as Mycoalert (not FDA approved).

3740....seems like alot of money BUT if you do any experiments at all....add up the cost of doing experiments on Positive cells:
Lab space costs
Post doc/Technician wages.
Consumable costs.
Costs for using specailised equipment.

AND you cannot publish any of these results.

Collaborators: Have they done the right testing at their end???????

Mycoplasma source: In the old days Foetal Calf serum was a major source but with filtration advances this is no longer the case.
The second major source is the tissue culture (TC) worker....have seperate lab coats for TC, always use gloves etc.
The third source is contaminated cells from other Institutes/Companies.
A fourth could be primary tissue if you are using the same cabinets for all TC work.

It has taken me many years to get our institutes down from 35% positive down to 0%. We do have new groups joining on a regular basis and this is the only time when we get positives again. These positives are quarantined so we never have any cross contamination of cell lines.

In our institutes we split TC up. We have designated rooms for:
Mycoplasma negative cells
Quarantine cells
Primary cells
Viral Manipulations.
Human Blood work.

This seperation again decreases the cross contamination risk of all adventiuos agents.

Kindest regards.

Uncle Rhombus.

-rhombus-

I am not criticizing anything but I recently had a similar problem and cried out for help on this forum - a few months ago. ALL my cultures were positive and with great determination and confidence about myself, I proved that those came from a particular vial of cells that I received from another researcher. Now believe me, I culture all my cells without antibiotics. That problem was a big setback upon me. Uncle Rhombus did post some useful info in the past.
What I did is wiped down all the affected incubators with bleach, then run the contracon _ High temperature cycle (for 24 hours!!) on incubators that was possible and then wiped down with 70% EtOH, the autoclaved the shelves in the incubator and tested for few passages with known cultures - well it was a hell!! But w think all that was unnecessary because like Uncle Rhombus says, mycoplasma / bacteria etc dont "JUMP" from one flask to other. I got massive mycoplasma contamination because, I use 293 cell to generate virus and then infect other cells with the virus - 293 was the culprit.
Things will go on. Don't worry. As for testing, yes it is expensive.
----- ----


We were just eliminating every possibility. We can't afford to send it out to another lab for testing. we are a completely new lab and have received 44 cell lines from different collaborators. (that's 44 * 85/test = 3740 just for testing one time....) (also, just wondering why a test has to be FDA approved? It's just for research right? MycoAlert is sensitive and tests a large number of cell lines, followed by a Hoescht stain it's pretty foolproof?)

we don't have a quarantine lab, we just have one tissue culture room with 2 hoods and 2 incubators so we try and keep everything seperate until they have passed the mycoplasma test.

I have been looking through and wondering where they are coming from but with cells from 3 different collaborators being thawed this time and all 5 cell lines being positive, it does look very much like we are the cause of it, SOMEWHERE in our lab. the common denominator is that the ones thawed were frozen down in our lab before the big clean.

So i'm grasping at straws and running our of ideas <_<

-SciCell-

OK, I've been reading a lot of the forum for the past few days.

But after lab meeting today, everyone seems to want a UV lamp although I never did have one in the previous lab.

The main problem is that we only have ONE tissue culture room. We do have separate hood for each incubator so it's the best I can do for now.

A collaborator (who is the scientist for my PI, who is a clinician) wants us to change the HEPA filter in the hood and fumigate the room. Is that necessary?

I wish we had a quarantine room with a quarantine incubator :(

-PostDocTrauma-

PostDocTrauma on Wed Mar 30 21:32:17 2011 said:


OK, I've been reading a lot of the forum for the past few days.

But after lab meeting today, everyone seems to want a UV lamp although I never did have one in the previous lab.

The main problem is that we only have ONE tissue culture room. We do have separate hood for each incubator so it's the best I can do for now.

A collaborator (who is the scientist for my PI, who is a clinician) wants us to change the HEPA filter in the hood and fumigate the room. Is that necessary?

I wish we had a quarantine room with a quarantine incubator :(


Dear Postdcotrauma,

You should only need to change the HEPA filter if it has been compromised i.e. has a hole in the filter. As the cabinet should be regularly tested under health and safety rules..for Containment and Ki testing.....this is probably not your problem.

You can fumigate the room if you can safely extract the formaldehyde/inactivate with charcoal filters. However if the room has been compromised by the environment then it will only be a short term remedy. It is more likely that the source is from a combination of things:
Bad aseptic practice.
A worker having a mycoplasma contamination.
Cells from non tested areas.

UV is not require for Tissue Culture......maybe 30-40 years ago it had a place, but with media prepared by companies, filtration methods for serum and contained cabinets rather than open work benches, it is no longer necessary.

I feel sorry that you are having a hard time....I have been very fortunate to work in pharmacueticals and in a university institute that has a total of 90 cabinets spread through 2 buildings....and quarantine facilties. This makes dealing with this problem very much easier.

I do hope you get sorted soon.

My kindest regards as usual.

Uncle Rhombus.

-rhombus-

could I please have details of the company that you send your testing out to? I'm going to try and convince the PI that it would be worth spending that money (but I've seen the budget for this year...and hope there's more money he hasn't told me about!)

-PostDocTrauma-
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