Hi Kavina,

As far as I know, mycoplasma are even quite hard to see with 20X, what you can do to be sure is make a mycoplasma detection test, either by PCR or luminiscent based test ( you will find several kits available on the web for this purpose) Once you confirm it is mycoplasma there are also specific antibiotics cocktails that you can use to eliminate them. For me, it is clear that your contamination is not bacteria but it could be is funghi, In that case you can treat the cells with anfotericin B and see if the contamination goes away. Another question, are you sure it is a contamination? Do the cells die? IF not could also be due to the kind of serum or media that you are using that for whatever reason causes these precipitation after long-term cultures??

This sounds like mycoplasma. They are not growing so strong, that the media gets turbid. I would definitely use a PCR based test to exclude the possibility or to confirm it.

Dear Sir, @ David Pacheu-Grau

I have used fresh lot of serum to avoid media component related toxicity. fungi like structures are cell debris due to this contamination . this culture dish was 90% confluent then it got this kind of infection and cells started forming clusters and eventually died. we are using myco related products but still couldn't get rid of it. these are seen in 20x. it is adherent to the dish and do not exhibit fungi characteristics. it spreads rapidly when the infected cultures are split further.

This sounds pretty much like mycoplasma. You will not see the infection (like any other bacteria infecting your media) and they are really hard to get rid of. My advice would be to get rid of all suspicious cells and media and to start over with fresh cells. You should also start testing your cells regulary as well as anything which comes in from anywhere else. Mycoplasma infection is more common than most people think.

Sometimes you can't get rid of mycoplasma, you just have to treat the cells regularly. I work with head and neck cancer lines that are frequently contaminated. It's just the nature of working with cells from the oral cavity and nearby areas. We have a separate incubator for cells known to be infected with mycoplasma, and whenever we get those cells out we treat them first. If you have a recurrent problem, I'd recommend treating as soon as you get them out or right before you make freeze backs.

Thank you @ Christian Praetorius and Elyse Walk. I will try treating the cultures or discard them. I think that's the only way :(

Christian's advice is the best. Just destroy the cultures. Most likely your stocks are also contaminated. I strongly suggest you obtain new cultures from a cell repository like ATCC. Mycoplasma contamination is most often due to poor technique and lack of quality controls such as routine mycoplasma contamination. Never, take cells from another lab. This is just asking for trouble. If you do get cells from another lab I suggest you quarantine them in a different incubator until you check them for mycoplasma or other types of contamination.

If it will be difficult to replace these cells lines I would recommend using MycoZap from Lonza. It find it works very well.

@Daniel: I wouldn't go that far to not accept cultures from other labs. But I would be very careful with it - assume that everything which is not tested negative, may be contaminated. This may sound paranoid on the first thought, but it can avoid a lot of trouble, especially when you analyze gene expression.

We have the luck to have an old incubator (which still works fine but is not as fancy) in another lab which is used when we get cultures from outside. When these are tested two times consecutively negative for mycoplasma, it can be moved into the main cell lab and we also make stock cultures of it.

One more thing: Mycloplasma is spreaded by improper working routines, not by having the flasks in the same incubator.

Regarding the treatment of mycoplasma, I agree that you should be really careful when you are receiving new cell lines, and you should control them before putting them together with your "safe" cell lines, but for me it is also a bit too much not to accept cells from other labs, sometimes you need to work with modified cell that can only be acquired from other labs. Moreover, although the contamination is a bit annoying to treat, usually it goes away after treatment. I would also recommend to make a regular control of mycoplasma contamination, every one or two months with the cell lines you are working with.

Alright i will start all over again with prevention techniques. i'll keep these suggested points in mind. though, i fear to get it again because we are sharing common room with other group members who are not ready to discard cultures. but i'll try my best.

In my experience the most common way to spread mycoplasma is via shared media bottles. Use your own media (and be very strict about it) and take out the amount of media you need into another tube (50ml or whatever you need). Keep the bottle with the rest of the media outside of the hood when working with cells. Discard any remaining media with that tube to avoid cross contamination. If you work with different cells lines, only work with one cell line at the time, desinfect your hood inbetween and avoid re-using the aliquoted media between different cell lines. These are relatively easy and still effective measures.

Alright , i'll be more careful and i'll keep individual media bottles for my work. Thank you