Mycoplasma contamination

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Guy Sovak
Guy Sovak's picture
Mycoplasma contamination

Hi all,
I got a big problem,
I got a Mycoplasma contamination in BHK cells,
2 question?
1- How do I get rid of Mycoplasma,
2- What is the best way to be sure that it is indeed Mycoplasma?
Guy

Richard Taylor
Richard Taylor's picture
If possible the answer really

If possible the answer really has to be throw everything away, disinfect and start again with fresh cells.

If you absolutely have to get into it then there are PCR detection kits, and reagents such as Mynox from http://www.minerva-biolabs.com/

omid
omid's picture
gsovak wrote:Hi all,

gsovak wrote:

Hi all,
I got a big problem,
I got a Mycoplasma contamination in BHK cells,
2 question?
1- How do I get rid of Mycoplasma,
2- What is the best way to be sure that it is indeed Mycoplasma?
Guy

Invitrogen has a kit to remove mycoplasma. If you have a very important cell line you can't throw away like some kind of rare hybridoma, you can try this:

PlasmocinThe Mycoplasma Removal Agent

Description

Elimination of Mycoplasma from Cell Culture
Plasmocin contains two newly developed bactericidal components strongly active against mycoplasma and related cell wall-less bacteria. Plasmocin can be used to both treat and prevent mycoplasma contamination in cell cultures.

Outstanding Efficiency
Plasmocin's potent activity comes from two unique bactericidal components. The first component acts on the protein synthesis machinery by interfering with ribosome translation. The second acts on the DNA replication by interfering with the replication fork. These two specific and separate targets are found only in mycoplasma and many other bacteria and are completely absent in eukaryotic cells.
Comparison of Plasmocin with the most common anti-mycoplasma agents.

No Recurrence of Mycoplasma Contamination
Plasmocin, in contrast to other anti-mycoplasma compounds, is active both on free mycoplasma as well as intracellular forms. This advantage is conferred by one component of Plasmocin which is actively transported into mammalian cells. It ensures that following treatment with Plasmocin a cell culture is not reinfected by mycoplasma released from intracellular compartments of infected cells.

Guy Sovak
Guy Sovak's picture
Thank you all for your

Thank you all for your answers.
I guess the best thing will be to start thoes new cell lines.
I looked into it and it seems that i indeed got some thing going there.
Guy

samm
samm's picture
Use Normocin, Plasmocin or

Use Normocin, Plasmocin or MRA for about three-four weeks, and split your cells as frequently as you can.
Also, do DAPI stains everytime and observe carefully - mycoplasma can be seen if they are there.
Finally, to confirm removal after four weeks, it might be best to have a PCR typing done.

Guy Sovak
Guy Sovak's picture
Thanks once again,

Thanks once again,
I did DAPI.
I will do as you say samm.
Guy

vanishing
vanishing's picture
I guess Richard Taylor's

I guess Richard Taylor's advice to throw away the cells is the best. If you are not really making stable cell lines, start from thawing cells.
And DO inform your colleagues who are using the same incubator, laminar flow, solutions to do the same if possible. Otherwise you will be passing them back and forth.

p.s.: how did you notice you had them?

BoricAcid
BoricAcid's picture
We have used Plasmocin on

We have used Plasmocin on mouse ES cells and come out successfully on the other side. Unfortunately, it is not definatively known if Plasmocin causes mutations.

It is easiest to throw everything away and disinfect (also make sure your tissue culture room has positive airflow) but it CAN be done!
Also, we used the PCR kit to check our other cell lines as well as to make sure the mycoplasma was truly gone.

BoricAcid

Guy Sovak
Guy Sovak's picture
I am happy for all of the

I am happy for all of the responces.
Eventualy what I did is
to Start from the begining.
I am not sure that all the treatments helped and that they didnt cause any mutation.
So,
I cleaned the incubators and started from step one.
Thanks to all
GUy

Elemental
Elemental's picture
 Dear gsovak,

 Dear gsovak,

Plasmapheresis is used to remove mycoplasma out of cells.
However, how is it removed from tissue.... Real human tissue.
I can't find the answer on the net.

elemental

Multiplexion
Multiplexion's picture
You can find a detailed

You can find a detailed protocol for Mycoplasma elimination at Multiplexion webpage.