Horrible mycoplasma contamination - (Jun/07/2007 )

Hi, everyone!
I'm totally new with tissue culture work. I just performed multiplex PCR screening on my new Vero cells, which my supervisor got them from another institution. They turned up to be mycoplasma positive.

My question are,
1. how effective is it to treat the contaminated cells with plasmocin (an anti-mycoplasma antibiotics)?
2. If I have successfully treated my cells, but the cells from others are mycoplasma contaminated, how high is the chance for my cells to get reinfection?
3. What should I do to minimize the mycoplasma infection to my cells, since others don't bother about this issue?
4. Will "isolation" of my flasks in a special container while incubating in the incubator help to minimize this nuisance?
5. If I ignore this nuisance contaminant in my cells, how serious will it affect my results? For your information, I need to use the cells to express my viral proteins and for protein-protein interacton studies.

Thanks a lot. I really appreciate your advices and suggestions.

Hi virus fan,

I am no expert on tissue culture either (have only been working with tissue culture for approximately 2 years) but this is what I think about your problem:

1. to treat cells with antibiotic can be successful, however mycoplasma contamination is extremely hard to detect at low levels, so you may think you have cured your cells but the infection has actually just gone below the limit of detection.

2. I'm not sure about transfer of infection between cell lines, but we do keep our known and suspected contaminated cells in a seperate incubator to those that are "clean". If you use correct aseptic technique and are very careful with your pipettes and aspiration/suction tubing, then I think theoretically the infection shouldn't be able to spread to a new plate.

3 and 4. You are right, many don't care about infection- too hard, or whatever. A collegue of mine got cells from someone else, they tested +ve, and when she informed the person who she got them from- the person refused to test their own cells and denied that they could have been contaminated before leaving the institute (dodgy scientist if you ask me!!!).
Like I said above, the best thing you can do is be extra careful with your aseptic technique, if possible quarantine the infected cells in a seperate incubator, and make sure you clean your hood really thoroughly after using it (although I'm sure you do that anyway )

5. Well its considered to be one of the most important tissue culture issues, but I'm not really sure how it will affect your results. Needless to say though- cells with an infection will obviously not behave the same as ones that are not infected.

Hope that helps a bit, and I will wait with interest for anyone who has more experience to expand on (and possibly correct ) my limited knowledge

Laura

Hi virus fan,

I am no expert on tissue culture either (have only been working with tissue culture for approximately 2 years) but this is what I think about your problem:

1. to treat cells with antibiotic can be successful, however mycoplasma contamination is extremely hard to detect at low levels, so you may think you have cured your cells but the infection has actually just gone below the limit of detection.

2. I'm not sure about transfer of infection between cell lines, but we do keep our known and suspected contaminated cells in a seperate incubator to those that are "clean". If you use correct aseptic technique and are very careful with your pipettes and aspiration/suction tubing, then I think theoretically the infection shouldn't be able to spread to a new plate.

3 and 4. You are right, many don't care about infection- too hard, or whatever. A collegue of mine got cells from someone else, they tested +ve, and when she informed the person who she got them from- the person refused to test their own cells and denied that they could have been contaminated before leaving the institute (dodgy scientist if you ask me!!!).
Like I said above, the best thing you can do is be extra careful with your aseptic technique, if possible quarantine the infected cells in a seperate incubator, and make sure you clean your hood really thoroughly after using it (although I'm sure you do that anyway )

5. Well its considered to be one of the most important tissue culture issues, but I'm not really sure how it will affect your results. Needless to say though- cells with an infection will obviously not behave the same as ones that are not infected.

Hope that helps a bit, and I will wait with interest for anyone who has more experience to expand on (and possibly correct ) my limited knowledge

Laura

Hi Rhombus,

Thanks for the extra info! I was wondering if you could let me know where to get information about the Hoescht test you mentioned.

Laura

I used plasmocin and it was very effective, I got rid of all the contamination.