I would back check all your media components and even the FBS as the DMSO is infected. Also, if you are culturing with antibiotics the probability of any contamination is that it will be antibiotic resistant. incubators, pipettes, biological safety cabinets all need to be looked at also if anyone else in the lab has infections in their cell lines?

Md Gulam Musawwir Khan, will do, thank you for your answer!

William Richard Webb, 

Nobody that I know of, no... The thing is, I am putting some compounds on the cells, i.e. gallic acid, which is dissolved in DMSO, that makes my curiosity even bigger. How can bacteria grow in DMSO with gallic acid, which produces ROS's and how can it still live when being put in the medium containing antibiotics??

antibiotic resistance, there are more than you think and if you use antibiotics any contamination will be antibiotic resistant.  try switching to gentamicin if you want to attempt to stop the infection other than trying to track down the point of infection such as fbs etc and in all probability the infection tends to come from the user. it might be worth decontaminating and starting fresh.

I would also filter any chemicals dissolved in DMSO using organic filters 0.2uM filter units. I dissolved chemicals and growth factors in both DMSO and water and would always filter prior to adding to media.

And/Or if you really want to find out the source of your contamination pipet some mL of each solution into a little cell culture flask and incubate for some days in the incubator. Check where it grows....

Thank You all for the replies! 

Well, it's not easy to track the source of the contamination, really, it could be from somebody else for what I know... Anyway, William, I did filter the chemicals dissolved in DMSO through the filter units. Maybe it could be the pre-contamination of the plates, I know that it should be sterile and all that, but what do you think? 

Friederike, thank you, a good idea, really, should have thought of that myself:))

in all honesty I would think it was operator induced infection. If no one else has infections then that would be my thoughts. Once you culture with antibiotics the likely hood is when/if you get infections they will be nasty ones due to the cell culture environment and selective pressures.

I would decontaminate the hoods and pipettes, new un-opened media and chemicals and also clean the incubators. it would be unlikely that the plates were contaminated if sealed but if you were using an already opened pack that is a possibility. As others have said I would place 3ml of media into T25 and culture for 24-72 hrs and see if it was in my base media and spiked media.

Yes, you're probably right. Will try to culture some media, what are you're thoughts about the type of media that needs to be cultured? Just DMEM or maybe the "Complete" medium (DMEM + 10% FBS + 1% P+S)?

As others said before: Sources of error 1) the incubator 2) your equipment 3) your reagents 4) your technique

In my experience and from what I've seen it has almost always been technique.

1) If it's incubator:

I would suggest plating FRESH brand new media w/o P/S as you want to directly compare to what your cells are being treated with and it's highly improbable that bacteria is going to grow in pure P/S. P/S may also slow the infection. It's possible that your FBS is contaminated too (depending on who aliquoted and how) so filter it if you can. Get the person who is least likely to contaminate the media to plate the media-if something grows then it's very likely the incubator. Sterilize the incubator.

2) If it's your equipment:

Do you find  you get contaminations more often with certain protocols, experiment, time etc.? This will tell you what equipment you should avoid. If you're sterilizing your tips etc then I don't think it would be equipment (unless you get equipment dirty while working).

3) your reagents

Whenever I worry about this, I plate first and foremost a blank (no cells) followed by a vehicle and keep them covered. Make sure you use autoclaved tubes to carry your reagents. Reagents should also go from sterile tubes to other sterile tubes. Check for contaminations.

4) If it's you:

Is absolutely everyone else having the same problem doing ~the same work in your lab? If not and the other alternatives are already crossed out, then it could be you. Wear a fresh lab coat when you work, double glove if you need to, avoid the grill at all costs, always keep the cells covered or mostly covered when working. 

Good luck, contaminations are always terrifying!

Thank you, Martino.

Well, everything is autoclaved prior putting it in the laminar flow cabinet. I also disinfect anything I put into it... I do not deny the possibility that it's my fault, despite all the carefulness and precision. What I've noticed that the contamination appears when I'm applying an assay to experiment, because when I culture the cells again and again, no contamination is seen in the flasks... 

Did you do a just reagents control? If your reagents are infected than something should grow and if your reagents are cheap then just replace them. My friend had a similar problem but he found moving slower minimized contaminations

Well, if the reagents would be contaminated, firstly, we would see it obviously from the first sight - the liquid wouldn't be transparent, it would be a bit cloudy from the bacteria in it and secondly, there would be a contamination in the flasks, where I culture cells. As for the chemicals which I use to perform the assays, yes, it could be the case, that's where my interest comes up, as I mentioned before, what kind of bacteria could live in an environment like DMSO + ROS producing chemical + media containing P/S:) 

If you see it under a specific experimental condition, perhaps an equipment or reagent associated with your assay  or your technique could be the source of contamination ? Have someone else try it out to see if the problem occurs again

Pen/Strep is only effective for 72 hrs at 37 deg C. Make sure you use a fresh aliquot. The pen/strep could be keeping a low level contamination in check in culture, because they are getting media changes as you passage them. If your assay is longer than 72 hrs, and your pen/strep is not freshly thawed...a low level contamination could show up.

Thank you for your answers!

Nancy, did not know that... Interesting:) Because all of my functional assays require approximately 3-4 days. So, as I understood, you would recommend to change the medium after 2 days on an outgoing assay?

If you can without affecting your assay, I would. If not, just make sure the Pen/Strep is thawed and added to your assay media right before plating the cells. Good luck, and let me know if this helps.

Will do. Thank you, Nancy, good luck to you too:)

Contamination control or check should be done at all stages beginning from the working surfaces, the safety cabinet, the reagents, the storage space (fridge where prepared media etc are kept when not in use), incubator etc. Ensure that the antibiotic is of good quality and is used at the right concentration; perhaps the contaminating bacteria are resistant to one or both of the antibiotics and you may need to change to another one e.g. gentamicin. Also ensure that the working space or culture room is sterilized regularly and access to it is limited restricted only the researchers involved.

Check if the composition of your medium is according to ATCC, and prepare all of your staff in sterile condition. Check also your laminar and incubator and most important water bath could be contaminated so I would suggest to clean it carefully. Also check pipette ( I would leave them under UV to have them sterile) and flasks sometimes  the material of flask is not good for some cell line. If not maybe you could have problem in your antibiotics stock, you could try to use a new one.

Good luck

Thank you for your answers!

After a lot of discussion here and also with my colleagues, we've probably found an answer to the contamination problem...

The thing is, when I'm applying my assays, I'm trying to observe and measure the viability, migration / invasion of the cancer cells affecting them with ROS - producing substances but also adding catalase, to see if it's the effect of H2O2 and what are the differences of cell specifications with and without catalase in the medium containing certain substances. I've written catalase in bold because we believe that it is the case for contamination. We weigh the catalase in the scales unsterile so I think that could be the main problem here...

Now I'm preparing an assay to test if a contamination appears in the cell culture adding just catalase to the medium, just DMSO to the medium and leave the cells unaffected by anything - just with the Complete medium and then observe the cells in the period of 4 days (because the contamination would appear approx. after 2 days)

What's your opinion on this? Is it a good idea?

Hello,

I've got rid of all of my used media constituents (DMEM, FBS, antibiotics) and changed into new ones. To my mind, pure DMSO should be toxic, that's why I was so surprised - not only the bacteria were live in medium containing DMSO, but also in the medium containing my research compounds, dissolved in DMSO, which were cytotoxic to my cells... So I've decided to take a new bottle of DMSO and filter it through your mentioned membrane filters before using it as a solvent. 

I couldn't save my cells from contamination though, so I had to cultivate some from the other flasks.

As a suggestion, of course, basic laboratory hygiene should be maintained, every reagent should be opened and closed in a sterile environment, the incubator should be cleaned every once in a while. Sterilize everything carefully before putting it into your sterile environment (ex.: laminar). As for the compound dissolved in DMSO, try doing it like I did, take a different bottle of DMSO and filter some into an experimental tube and try to dissolve your compound in it. And then try to check if it has bacteria in it - put some of your old solution into one well and the new one into other, check after 24hrs if it's contaminated. 

Hope this helps