I have been frustrating with certain contamination issues for a while, and strangely I kept having the same contamination problems (yellow color and round shapes). The media was not cloudy by eye (only can see them when they became large), but there are tiny dots that are usually floating or settle at the bottom of the culture dish. It keeps showing up every 2-3months after we toss cells, decontaminate the incubator, and defrost new cells. Any suggestions to help us to overcome this? Thank you so much in advance!!
I witnessed this in my lab many times, as seen from your pics I think those are might be fungus contamination which is quite a general contamination problem.
Can you observe large or small white-grey dots floating in your medium?
In this case, throw the cells, renew culture materials (trypsin, PBS, medium),
Sterilize the inside of the incubator with ethanol (70%).
If you suspect from medium, pour some medium into an empty petri dish and wait for 48 hours to observe whether there is any contamination.
If you still have these in your culture, you may increase (use above 1% of medium volume) the concentration of the antibacterial-antifungal reagent in the culture medium (generally preferred agent is penicillin-streptomycin with 1%).
You sterilized everything, increased the penicillin-streptomycin but still have contamination with new cell? Then you can suspect from the cell stock in the freezer or liquid nitrogen tank because sometimes we freeze the cells as infested with another organism unknowingly.
Hello Mahmet,
Thank you so much for your suggestions! It's very strange that I never see any suspect things on my media bottle and not all of my cell plates are contaminated even though I used the same media stock. If the media is the source of the contamination then, it should contaminate all cells...? Also, I pour some media into an empty dish and keep it in the incubator for 3-4 days but nothing grew (just look like fresh media). However, this contamination is really randomly happened. I never tried using a higher concentration of pen-strep (I also use 1% in general) so it's a definitely good point to know!!!
Whenever I saw this contamination, I always toss all contaminated cells, start making new media & PBS, cleaned a water bath, incubator, and our BSC cabinet as well. I even bleached the walls and floor... I tried to clean everything but it keeps popping up in a random time manner.
I also suspect our original frozen cell stock. If that's the case, then I suspect I'm supposed to see them within 3-4 days after defrosting cells. However, I never see them for 2-3 months even though I passaged them many times...
I will definitely increase the pen-strep concentration.
Thank you so much again for your help and very helpful suggestions!! I really appreciate it!
Ok, I am happy to hear that I am helpful, but be careful, increasing pen-strep concentration can harm the cells or decrease the rate of growth of your cells. If you have enough source, I suggest culture your cells in different concentrations of pen-strep in different flasks.
I hope everything will be ok with your studies.
Hi Rosemary,
I think I would have to agree with Mehmet, that does seem like fungus contamination. Its source could be variable, so, in addition to what Mehmet already recommended you, I am going to give you some pieces of advice based on Freshney (2010).
As you described your issue, it seems like a repeated contamination, so the first thing I would suspect is that contamination comes from the reagents employed or the cell line.
I see that you already poured some fresh media in an empty dish and incubated it, and nothing grew. In our lab, what we do to test media contamination is that we plate it in a petri dish with plate count agar medium (PCA) or potato dextrose agar medium (PDA) to see if there are any bacterial or fungi contamination, respectively. I think you could try this instead of just an empty dish, and discard it if any colonies grow on either plate.
Alternatively, sometimes contamination comes from the simplest things, such as an overcrowded hood or lab, room air quality or the use of the same laboratory coat on different laboratories. Perhaps you could evaluate if this could be an issue. I am pretty sure you have lots of experience with aseptic technique, but constantly reviewing and enhancing aseptic technique is critical for avoiding contamination.
If contamination persists, probably the most adequate thing to do is to discard the cell line and ensure future imported cell lines go through quarantine. As I saw you are planning to increase pen-strep concentration, however, sometimes increasing antibiotics concentration could be unsuccessful or lead to the development of an antibiotic-resistant microorganism. Therefore, I would not recommend this unless it is the only alternative, so please be careful.
I would like to know if any of these tips help you with your research. Have a good day! :)
-Josué
References:
Freshney, R. (2010). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. Ed Wiley - Liss. USA.
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