I think I know what you are looking at.  When I was doing cell culture in 96-well plates years ago I would see what you are describing.  I found that you would see the black dots even if you plated out just fetal calf serum and incubated that for a period of time especially when using round bottom plates.  I don't recall how long but probably a week.  I used this method to screen different lots of serum to find one that didn't have the black dots and I would use that for my cell cloning work.

Sucks, that's likely bacteria contamination.  Are you filtering your media with a 0.22um filter before use?  Would it be possible to get a fresh lot of cells then use another groups equipment for a day or two to see if you can at least get an uninfected culture started?  You may need to look up decontamination protocols for your incubator as just washing the visible surfaces may not get at all the nooks and crannies where bacteria can hide.  Getting rid of a stubborn bacterial contamination is a pain.  Good luck!

The best way to solve this problem is throw your contaminated cells away. The cell line is contaminated with MYCOPLASMA. You can buy a kit to test your sample. You can buy chemical to kill mycoplasma but it can not remove completely the mycoplasma. Thaw new cells, remove old chemical, and sterile the cell culture bench to remove completely mycoplasma.

I've tried 2 new cell lines (sourced from ECACC) all media is bought in sterile filtered from Sigma Aldrich. my incubator has a self sterilising process which I've utilised between throwing cell cultures out and ordering a new batch in. My hood have UV sterilisation and I absolutely drench the thing in IMS and clean weekly with virkon. I'm pretty thorough when it comes to cleaning my lab routinely. 

I was under the impression you couldn't see mycoplasma under the microscope and these dots are pretty visible.. They look like this ( please see link)

https://youtu.be/lo96fcXfcVs

I watched the youtube. What cell line are you growing now?

In my case when I worked with NIH3T3 cells and I saw tiny black dot around cells after new split. For the second day, somehow these black dots I could not detected anymore or hardly detected. My explanation was the cells got stressed and died due to the trypsin treatment step even though you add media with FBS to neutralize afterwards. Moreover, if you grow cells with low intensity, you can observe tiny dots around the media (not apply for all cell types).

In your case, I just think that the cells enter to the apoptosis cycle. What do you think?

Yep, that's definitely a low level bacterial contamination.  You're right that you can't see mycoplasma, even in a microscope, which is why you need a kit to detect it.  While I have seen some cell lines that produce large amounts of debris, debris doesn't move around on its own, so this is not just due to cells dying.  A few things to try:  First, get an aliquot of a cell line from another group, or from your own stocks, that you KNOW is uncontaminated.  Bring that up in all your old equipment, media everything.   Check and see if it's contaminated.  Second put a plate of just media, no cells, in your incubator and see if it comes out contaminated after a day or two.  Third, do the same in someone else's incubator using their media vs yours.  Third, consider another source for cells.  Fourth, I realize that companies always say their media is sterile filtered but I filter my media along with my FBS anyways.  This is a good habit to get in to and if all of your other aseptic techniques are good, should prevent needing antibiotic at all.  Finally, you should try all of the above steps with and without antibiotic to assess bacterial growth as even a low level contamination will go out of control without antibiotic.  You may also need to consider everything you use on your cells as suspect: plates, flasks, PBS, pipette tips, hood, everything.  Start with everything fresh, preferably from different sources or from a lab without contamination and try to get to a point where you have no contamination at all, then work backwards from there to pinpoint the source.

Hi Rose,

I think most of the precautions have been pointed to you, however recurrent infections are due to your handling and also the sterile bench. Always, wipe the glas surface closest to your face by 70% ethanol spray before you deal with the cell-culturing, spray your gloves and spray to the new empty culture plastic-ware required for the day inside the hood, then switch off the UV-light. Take the cold media to new culture tube(s) and to the falcon(s) needed for the moment, put back the cold medium into the refrigerator. Now, you leave the tubes in the hood for 15-30 min, meanwhile you look to your cell cultures or your do other calculations in the cell room ....etc. Then after you handle normally with the cells for the next passage according to your routines. These essential steps are definitely minimizing contamination source(s). I hope that you overcome with this problem soon ....

Good Luck ....

// Beston

Since the black dots appear to be moving, they're bacteria in all likelihood, most probably Mycoplasma. I have seen this before and the way I took care of it was to treat the culture with 20ug/mL of cyclosporin, replaced every 2-3 days, till the bacteria died out. Getting new cells from ECACC would not help if their entire stock is contaminated with Mycoplasma, which is not that uncommon. You could talk to the technical support department of ECACC and tell them about the problem. I don't think this is an issue of you doing something wrong or there being a source of contamination in your incubator/ reagents/lab. Does anyone else using a different cell line (and unshared media) have the same problem as you? If that's not the case, it has to be the Mycoplasma contamination at source.

Hi Rose,

I agree with all possibilities above, just want to add one more possiblity from experience. No amount of meticulous work and adhering to SOPs helped us until we figured out that source of our contamination was liquid N2.

As you may well know N2 will end up inside some cryovials and some of the dirty buggers can leak out. Although frozen, some happily survived (might be spores as someone mentioned above), and kept contaminating everything. Of course it all gets a lot worse when contaminating bacteria are already pre-selected with antibiotics and therefore resistant.

Just one more possibility to consider.

Good luck.

I really appreciate all of your comments! This is such a massive help! Thank you!

I have not shared the lab as I am currently the only employee working with cells at the moment and just the PC12 cell line. 

I've tried using TSA plates to source contamination but I couldn't get anything to grow? We are a tiny start up CRO and so i don't have the facilities to identify the contamination in order to treat it. so any suggestions would be greatly appreciated.  

The number of black dots have demished over the second passage and have not grown back quickly this time, struggling to actually find some now. Where as before they quickly turned the media cloudy and deminished the number of viable cells. 

I have sourced everything new, and have also now sourced 0.2 um syringe filters to filter everything myself, as a precaution as well as buying apparently sterile filtered in. 

As it is a newly set up lab for a tiny CRO and unfortunately we don't have old stocks that we can guarantee are sterile as my peers before me ran everything with pen/strep which means it could have also had an underlying low level contamination.

I had sourced cells from a different company that had been run without pen strep as a control but upon picking the cells up from their lab and bringing them quickly here I checked them under the microscope and they appeared to have the same problem. So that ruled out using their lab also. 

Its definitely not fungal. I had ruled out mycoplasma due to being able to see them down the microscope, however I have invested in some mycoplasma detection reagents to check everything I have.

I'm unable to spray whilst the UV light is on as the nature of the hood shuts the screen with the UV light on and then reopens after the cycle, where i then cover everything in IMS and spray everything i can into the hood for that cycle of work. which I then allow 15 minutes to dry and allow the fumes do their work.

I would love to explore the possibility of a contaminated LN2 dewer, but how do you go about decontaminating the dewer? 

Hi Rose,  

If you are seeing your media go cloudy, discolour and your cell viability drop then bacteria is the most probable cause.  As you have replaced your cells already I would suggest performing sterility testing on the cells you have, you can outsource this to companies such as ECACC which is a lot easier and cheaper than doing it yourself.  I would also suggest a mycoplasma test at the same time, ECACC can do this too or there is a company called Mycoplasma Experience which is also good and a little less expensive than ECACC for this.  Just because you can see it doesn't mean its not mycoplasma unfortunately, you often can't see mycoplasma but sometimes you can.

Black dots don't always mean contamination though, it is possible for cells to shed protein / debris especially if they are highly mutated tumour cells and this can become more obvious if the cells are stressed, for example after freezing/thawing.  This can look like the wiggling back dots of bacteria, but won't turn your media cloudy.  This isn't particularly common, but can happen.  

If you are hoping to filter sterilise materials, I would suggest using a 0.1 µm filter under relatively low vacuum pressure as mycoplasma can get through a 0.2 µm filter, they can also get through a 0.1 µm filter under high pressure.

Contamination directly from the nitrogen dewar is unlikely, this only really happens if you have a contaminated cell bank in the liquid phase with a vial which is not closed properly, contamination then happens when you thaw the 'clean' cell line though poor aseptic technique on revival.  If you are a new company, with a new dewar I would explore other options first.

If you end up replacing things again try using different supplier for everything, you can get the cells from ATCC, media and FBS from Life Tech etc.  Can you get a different cell line all together?  The other thing to check is that you are using filter capped culture flasks and not leaving the lid open? And obviously, check your aseptic technique.

I hope you get it sorted, this kind of thing is a nightmare!

Charlie

https://www.phe-culturecollections.org.uk/services/sterilitytesting/sterilitytesting.aspx

http://www.mycoplasma-exp.com/

Dear Rose,

Are you using FBS for you cell cultures? As it is also recommended to filter it, as potential source of contamination. This is what happened couple o times in our lab, before we managed to identify FBS as a victim, and now we filter it all the time.

Good luck

Aleksandra

I think it is a Mycoplasma

Thanks charlie thats really helpful! will suggest these options to my boss as its something i haven't explored. thank you for your time.  

ATCC however are out of the question though as they take 3 weeks to ship and are double the price unless you know of a UK distributor?

Hello, I think we have a very similar problem. Did you manage to sort out yours? Can these be Achromobacter? Thanks very much in advance. 

Hello.. there was absolutely nothing wrong with my cells! I tested them for absolutely everything I knew how to and all came back clean. I put it down to brownian motion as I was using a microscope that was a tad excessive for cell culture. 

I spent so much time and money trying to work out what these tiny dots were, came done to inexperience on mine and my supervisors part. 

Thanks very much Rose. I was suspicious about brownian motion as well in the beginning but these flipping little things kept proliferating (very slowly compared to any kind of bacteria I have seen) and they came positive when stained with Hoechst :/ Glad that yours were false alarm (sorry though as it costed a lot it seems). Many thanks again. 

your welcome! good luck and hope you get them sorted soon!