Washing the cells with PBS won't fix contamination unfortunately. But that is a standard procedure used to care for cells generally (they need new media every few days to remove waste products and provide more nutrients for growth). PBS is a common cause of contamination though. You should always make up a sterile batch of PBS, only use it for a specific cell-line, and never open it outside of the hood to prevent contamination.

It sounds as though your original culture (i.e. the frozen down one) might be contaminated. If you have frozen vials from a different cell line, try waking them up and seeing if the contamination reappears. If not, then you can be almost 100% certain that it's not any of your lab gear that is contaminated; and your aseptic technique is not at fault. Before doing that however, sterilize all of your pipettes and change the filter inside; then get a new box of sterilized tips to work with. Pipettes (especially serological pipettes) can get contaminated quite easily, especially if you accidentally suck up too much media too fast. Also make up new PBS or whatever you are using to wash the cells. Hope that helps, contamination is a pain!

*NOTE: Unfortunately, if your original culture vials are contaminated, then you'll have to purchase a brand new culture for that cell-line. There's no reliable way of removing bacterial or viral contamination from a culture without compromising the results of your experiments. Because you can't know whether the contaminating organisms have influenced the genotype of the cell-line.

Dear Joanna, thanks for your complete explanation. We have testes two completely different cell lines L929(mice fibroblast) and MCF-7(breast cancer). they all get contaminated.

I have worked with either samplers or pipettes! so I think there will be another source for contamination.

I'm worried the creature may treat those working in the lab :s

in case of viruses, how can I completely destroy them?

Hello, dear researcher .I think is better you change media after 2-3 days. 

Dear Malihe, I have Totally changed the media. I made fresh media and filtered it. yet didn't work

Hi, do you test your media before use? Are you using new tips? And your falcon tubes are new? 

Do you have another vial of these cells? From another batch?

Well it's definitely diagnostic to know that all the cell lines are getting contaminated. If the samplers and pipettes haven't been sterilized, then that would be the best thing to do next. And replace any filters in your pipettes. From the picture you posted, it looks more like a bacterial contamination (although I'm no expert in recognising microbes). Like I said in my previous post, contaminated cultures should be disposed of. If it is viral you won't know unless you are doing PCR or some other test which would pick up genetic contamination. You can't see viral contamination under a cell culture microscope.

It seems most likely that your issue is with aseptic technique. This does not bode well for the remaining frozen vials of cells.  Make sure that you're doing the following whilst handling the cultures:

1) Keep the fan in the cell culture hood on all the time, to suck up all the potential bacteria that might drift in. If you aren't using a cell culture hood, then it's no surprise that contamination is an issue.

2) Spray down EVERYTHING that enters the  hood with 70% ethanol (including your gloved hands if you remove them from the hood and touch something outside it). The only exception to this is the culture flasks themselves (if you spray them with ethanol it will get in through the vents and they will die).

3) Any substance you plan to use in your cultures needs to be sterile. Cell culture media comes sterile, so it should only ever be opened in the cell culture hood. Never double-dip your pipette into the media; switch tips every time to avoid contaminating the media.

4) Whilst working with the cultures (e.g. changing media, etc.) are you disposing of waste from the cultures in a bin outside the hood? If so, don't do that. Instead, get a plastic container (like an old cell media container), spray it with ethanol and use it as a little bin inside the hood. Then you have reduced your chances of contaminating the pipette on the outside of the hood.

Both your samplers and pipettes could be contaminated, so I wouldn't rule out that as a potential source. Especially if they are shared, and haven't been sterilized in awhile. I'm not sure what this means "I'm worried the creature may treat those working in the lab" - I'm guessing you are worried that people working in the lab will be infected with the contaminating bacteria? That won't be a problem unless people don't wear gloves, touch their faces in the lab, and do other things which they shouldn't be doing in a PC2 lab.

Hello.You should wash your flask after 2-3 days by PBS and change its media. I had similar case.

Good luck.

Washing the cells with PBS won't fix contamination unfortunately. But that is a standard procedure used to care for cells generally (they need new media every few days to remove waste products and provide more nutrients for growth). PBS is a common cause of contamination though. You should always make up a sterile batch of PBS, only use it for a specific cell-line, and never open it outside of the hood to prevent contamination.

Dear all,

thanks for the very detailed answers.

unfortunately, I had done all the instructions and yet cells gonna die. I completely work under a laminar hood, I spray ethanol 70% on everything before taking it under the hood, I've made fresh new media and I've filtered them all. and absolutely I had every piece of dishes sterilized with a autoclave. the contamination yet remains.

I also put sterilized  nutrient agar dishes in different parts of the lab to find out where the contamination comes from. the ones under the hood, in the fridge, in the incubator and in the autoclave remained non contaminated. only the one in which I poured drops of culture media over the cells(from the flask) showed signs of bacteria.

I am wondering whether the contamination is from -80 degree fridge.(the one we keep all cell lines in). can bacteria survive in -80 temperature?

I have a few additional questions, just to make sure;

I hope that you find a solution for the contamination issue.We are experiencing the same issue in our lab and we were not able to rule out the source of contamination in spite of doing all the sterilisation techniques including autoclaving, U.V light and many other.

Hace you checked for presence of mycoplasma? they are difficult to detect and hard to get trough.

Hi, for how many days do you leave those plates with agar nutrient opened at your culture room? I want to do this to see if maybe I have a source of contamination.

Oh, and was your problem resolved?

Thanks!!

Dear Jordana, Hi

sorry for the long delay. nutrient agar becomes contaminate after a day if there is a source of contamination

our problem is solved:)

the problem was with the air filter of the clean room(the laminar hood was ok). we changed the filter and installed a UV lamp in the clean room