5 replies to this topic

[PostDocTrauma]

Hey,

Earlier in the year the new lab I started working in accidentally imported some myco+ cells. These came from a collaborator's lab that promised us they did mycoplasma testing but their testing method involved growing cultures for 3-5 days and discarding it.

So most of the cells we received tested +ve from them and we were so sure they should be negative. We started blaming the supplier of our tests (Lonza) but in the end we sorted it out.

We cleaned EVERYTHING thoroughly and installed some UV lamps. I made sure everyone used everything once to prevent cross contamination. This was fine for a few months.

Except now in our regular testing, we have mycoplasma again :-( :-(

We have cleaned everything again (we are all so sick of cleaning out TC lab) but I'm wondering if it's worth paying a company to fumigate the whole lab so we get rid of it completely?

The main problem is we have no quarantine area, we have 3 TC hoods but only 2 incubators. And these are all in the same room. I've been promised another TC lab but we won't be able to get it for another year....

Any help/advice/hugs anyone?

[bob1]

It sounds like you are doing the right sort of things to get rid of the contamination.  Perhaps there is some hidden source (like lab coats) that are coming into the lab contaminated.

Have you checked that it isn't in the cell stocks?  - the tests have detection limits but a few day's growth should be enough to detect basal levels.

[nightingale]

Hello,

have u tried using :1,1,2 trichlorotrifluoroethane ???

we are having a stock, that was tested positive for Mycoplasma ...
and came to this procedure , ATCC applied, & are to try it ..

here is the procedure :-

1. Take 4.0 mL of mycoplasma positive viral stock and freeze/thaw twice.  

2. Dispense the stock into four 1.5 mL microcentrifuge tubes.

3. Centrifuge at 14,000 rpm for 30 minutes at 4°C.

4. Transfer the supernatants to a 15 mL conical tube (save 1 mL as a positive control).

5. Add an equal amount of cold 1,1,2 Trichlorotrifluoroethane into the tube.  

6. Vortex at maximum speed for 3 x 1 minute.

7. Centrifuge at 1000 rpm for 15 minute at 4°C.

8. Take the upper layer out of the tube carefully. Consider this your "treated" suspension and use for passage to grow more mycoplasma-free culture. Note that this treated suspension must be tested by PCR or another method to verify that it is mycoplasma-free. It may take 2-3 passages to be mycoplasma-free.

  
hope it will work for u ... if so, please do tell us.

all the best,
nightingale

[bob1]

That might work for viruses, but won't work for the cells the mycoplasma actually grow in.  The vortexing alone would probably kill them, and the pelleting step would remove the cells from the culture.

[PostDocTrauma]

bob1, on 10 September 2011 - 06:54 PM, said:

It sounds like you are doing the right sort of things to get rid of the contamination.  Perhaps there is some hidden source (like lab coats) that are coming into the lab contaminated.

Have you checked that it isn't in the cell stocks?  - the tests have detection limits but a few day's growth should be enough to detect basal levels.

just dumped all the lab coats to the wash room.

we're now thinking about pipettes so have started using filter tips in everything. But hopefully the new lot of cells to be thawed will be clean.

[briguy7]

Do you have enough space where you could designate one of your hoods to only work with the untested stocks/materials while you are testing your stocks and materials?  Anything you can do to keep the cells/materials that you know are clean away from the ones you don't know about would be good.  I would also toss your DMSO or freezing media and at least sterile filter all your medias.  In case you haven't thought of it, I would advise against using any antibiotic--it would just slow your detection process anyway.  Even if you can't have a separate TC room, you can do your best to keep everything separate as possible.  Another thing that I used to always forget about, really do a good job of cleaning your water bath you use to warm your media.  I agree that going with all filter tips is the right idea--maybe even autoclaving your pipettes and pipette aid, if possible.