I don't think that is bacteria specially since it doesn't grow using LB media. They look fungal since you can see some budding but I'm no expert in micology. Probably your Hek stock was contaminated before freezing. Have you tried other batch of frozen HEK cells? Maybe you could ask another lab for a cell sample since getting rid of fungi can be nearly impossible and Hek cells are quite easy to find next door.

I am more worried about the source of the contamination, since it is in the FBS (ours and the one that we borrowed). Now even if I borrow another aliquot, how do I make sure this does not happen again when the FBS is still carrying the contaminant. I have like 10 bottles of serum and cant afford to throw them away. 

Since you tried two different sources of FBS the contamination could in fact be in your cell culture. Have tried to add some medium on a well (no cells and no PenStrep) and check it after a few days? I always like to filter my medium when I add the FBS, it's easier to filter a 5% or 10% solution than the 100% FBS stock. The problem is what kind of experiment you will perform afterwards since fungi can release molecules that activate cells. If your closed FBS stock is contaminated it can be a manufacturer problem and your local distributor will replace it.

I tired it. Thats how I found out the contamination is from FBS. I opened a new bottle of media and a new bottle of FBS. Put some media, FBS, media + FBS in 24 well plate and I saw the contamination in all but the media. So, if the contamination is from the lab/hood/pipettes, wont the media also be contaminated ? Also, when I borrowed the FBS and put it in a six well plate, I could see them straightaway under a microscope even without incubation. I have requested the other lab to do the same and see if they see anything. Maybe that will rule out something. I am doing transfection, IF experiments with these cells. They seem to behave fine with all these but for the contaminants stained with DAPI. 

It doesn't seem to be you lab equipment. The wierd part is that the FBS from another lab has the same contaminant. Do you have 0.22um filters in your lab? Try to filter your complete medium. I believe you won't have any problem if you are not transfecting immune related receptors.

Thanks ! That is a good idea, I was also thinking the same to filter the media. My proteins of interest is related to DNA repair. 

Dear Shivasankari,

1. Filter  DMEM+10%FBS+pen-strep complete media with 0.22 micron membrane before using. You should never use unfiltered FBS in media. It will evoke contamination always. 

2. It is not bacteria as ruled out by you. Bacteria tends to stick to the surface of the plate and appear as black dots at higher magnification. Also, from the images you provided, it doesn't look like budding yeast. Budding yeast will distinctly form budding structures and make a chain, which is easily visualised under light microscope. Moreover, if it is any kind of fungi, the media would have turned yellow and turbid rapidly. It is the first indication of yeast contamination. 

3. That leaves us with mycoplasma, which shouldn't be visible under 40X. But as it is not showing similarity to any other type of known contaminants, let us assume it to be mycoplasma. In that case, you should filter your media (all included) using a 0.1 micron membrane filter to avoid any chances of contamination. Although I haven't used but, one of my friends have used Plasmocin from Invivogen. It is very efficient in removing contamination. You can read about it in the shared link.

If not, you first should filter your complete media with a 0.22 or 0.1 micron filter before using. I think that will help you.  

http://www.invivogen.com/plasmocin

I really don't think that is mycoplasma. These contaminants are too big to be mycoplasma. They look like fungi conidia.

Thank you all. Yes, I don't think it is myoplasma either. Myco testing is also in the cards but not for this reason though. I just cleaned out everything again and I am going to heat inactivate my serum and filter the media+serum+Penstrep and use it. I hope this helps or at least eliminates some. Will keep you posted.

I'm no expert in microbiology, but, contrary to what others may say, it does look like bacteria. But, you would expect it to grow really fast if that were the case.

Try to wipe the outer surfaces of your flasks/growing chambers and see if you still see the baceteria-like structures.

Also, if the serum is not heat-inactivated, how can you be sure its sterile? Because, if its not, it would make sense that a microbial colony could grow especially if there are no antibiotics being used.

Maybe, if you are desperate and/or willing to experiment with antibiotics, you can always try a a tetracycline antibiotic to remove a certain pesky strain that may be the culprit bacteria in your culture.

Thanks Prashanth ! I tried wiping the outer surface of the flasks and also the microscope. Not of much help. Serum comes sterile and the heat inactivation is to inactivate the complement system which might affect some applications. This has been done in the lab routinely. If nothing works, I will switch antibiotics. I am just worried about the stress on my cells. 

Hello all ! I think I just found out what that could be. We have strong suspicions that those are liposomal DNA complexes, because when I did an experiment with LPEI only, LPEI + DNA, DNA only, No LPEI/DNA,  I found those round structures appearing in LPEI only, LPEI + DNA and not the rest. Lipofectamine 3000 also gave the same results. I found the round sturctures in coverslips (without cells) incubated with the transfection mixture. This also explains 1) No change in the media appearance/pH 2) No effect of the cells 3) Staining positive for DAPI 4) Rapid increase in them within the 6 hours of transfection. Also, the amount of these structures increased with increasing concentration of DNA. Regarding the structures that I saw in the media/FBS, I think it is some random particle or a protein ppt. A question that is still open is why do these form ? Why hasn't the lab seen this before ? I am continuing to find answers for this. But atleast I dont have to worry about it being a biological contaminant and can stop getting paranoid about the cleanliness of the lab. Thanks everyone for the inputs !

Hi Shivasankari,

My lab is also having a very similar problem as yours. We tried to take out some drops of the contaminated samples and culture them in LB and YPD plates to see whether they are bacteria or fungi. However, it did not show anything.

We also tried to add transfection reagent only, transfection reagent + DNA, and DNA only to HEK cells coverslips. We saw the samples that were added with transfection reagents developed round dots.

May I ask if you have any suggestions of dealing with this problem? The transfection reagents that i used were Xtreme HP and Xtreme 9.

Thank you!

Hi Le, 

      Exactly, my problem. One thing I could conclude is that they are transfection complexes. Reasons :

1. They stained with DAPI, which means they have DNA in them. lipo complexes have DNA in them.

2. In spite of having no cells at all, adding the mix on the coverslips with OPTIMEM gave us those round spots again. 

3. They are almost uniformly shaped, which makes me convince they could be lipo particles. 

4. No change in the turbidity or color of the medium; did not grow in LB, post transfection they appeared to be many many many fold increased and the cells grew well - all these suggest that it is neither bacteria nor fungi. 

5. Myco will not be visible under light microscope under such low magnification, so no myco. 

6. They increased with increasing DNA conc in the mix. 

7. Lipo 3000 (company made) and LPEI (lab made) both gave me the same results. 

     I havent found out a way to eliminate them, but one way to minimise would be to use more cells, so have less surface available for the mix and also to saturate the lipo mixes. 

Hope this helps. 

Hi Shivasankari,

Thanks so much for sharing with me your problem with the transfection and the solution. We have ver similar problem to yours, and we found that by using less amount of transfection mixture (DNA+OPTI-MEM+transfection reagent), the "contaminated particles" reduced significantly. Moreover, when we switched to Xtreme 9 transfection reagent, the particles also reduced significantly (we used Lipo 3000 and Xtreme HP reagents before).

Hi Trieu Le, Glad to know that you were able to sort out your problems and that I was of some help in it. All the Best. 

Two years later: I'm glad things worked out.

Wanted to point some stuff out for future readers:

1. I mentioned "wiping down" exterior parts of lab-ware. By that, I am assuming with 70% (v/v) ethanol/water is used to sterilize it without getting any on the inside. Handling sterile is key (though it may be an unsaid assumption).

2. You are right that heat inactivation is intended to inactivate the compliment system (and other enzymes (e.g., endonucleases)). However, the process (where protocols varying from heating serum to 56°C-60°C or -60°C-80°C for 20 min), cause denaturation, which tends to happen at > 43°C in mammalian proteins... Keeping in mind that prokaryotes too have proteins that can denature, heat inactivation may inadvertently inactivate enzymes and sterilize the serum. This matters because serum can be a rich source of nutrients for not only your cells of interest but also contaminating cells.

3. Those structures you mentioned are could be a non-living (e.g., precipitate of coalesced protein, or some complex mish-mash of biomolecules)... Or, contaminating living thing(s) (For example, it could theoretically be a contaminating bacteria that also is transfected inadvertently and uses DNA as a source of material as it replicates...that's just one possibility).

In any case, happy to hear you figured out a solution. Someone mentioned filtering, which was a great idea as well!