I have been culturing a primary culture of chondrocyte that isolated from human OA cartilage. I did this primary culture for several times and i never got a problem in my previous isolation. But this's the first time I got a cell culture problem. There were some black dots in my chondocyte culture. These thing are uneven shape and size. No active movement. But I am still confuse, is it an contamination or cell debris only ?
Here I attached some pict. Please give me some feedback and advices to handle the problem.
Just a short comment on distinguishing between cell debris and contamination:
1. cell debris usually can be eliminated by washing the cells with buffer while contamination will remain.
2. the medium color with debris does not change (pink/salmon) while contamination results in acidification of the medium and the color turns yellow.
3. hazy or cloudy medium always means contamination by microorganisms.
you may try with your new primary to switch to new antibiotics: eg. from penicillin/streptomycin mix to gentamycin. We have good experience with plasmocin, it is effective against bacteria and in addition against mycoplasma.
Dear Adrian Syawaluddin Siregar ,
I think it may be a contamination by fungi. Long time ago, I got a similar shape and well and truly a contamination by a fungus.Do you use gloves while subculturing cells?
Yes, I did it. How can I know whether this is fungus or not ? Did you test with some assay for your contaminated cell culture ?
If its possible you can add some Amphotericin B cell culture grade to the cells(as instructed on label), else take some cells out from your original flask and add it to some Terrific broth and keep in 37C incubator (add 0.5ml cells in 2-3ml TB), any contaminating organisms will love the TB and will be a confirmation,
If possible start fresh, if not possible, try spinning down and re-suspend in fresh media, joys of cell culture are not easy at times, best wishes
Thank you Shubhashish for your advices. I'll remember it for next isolation. I've got rid of the cells. Fortunately, I can still get new sample.
I dnt know exctly what your work is all about ie if it is important to isolate or identify contaminants if not just approach the black spots as contaminants.I av worked with acanthamoeba cells isolated from human retina and that looks like a typical contamination from either fungi or bacteria, which i ecounter sometimes.JUST discard the cell do not work with them and review your aseptic techniques while culturing. Cheers
If you're not sure you can fix and DAPI stain some of your cells. Mycoplasma or bacteria will show up as very small dots on your cells (which will be large nuclei stained) under a fluorescent microscope. Fungus is usually long strands under a light microscope with phase contrast. Black dots are usually mycoplasma or debris. Bacteria will turn the culture media cloudy. If you need to keep the cells you could always wash the cells briefly with PBS and 5 x antibiotics concentration and throw in an anti-mycotic such as Biomyc, Mycorazor or Ciproflaxin. Once established though mycoplasma is hard to get rid of.
At first time, after I collected some info from another RG disscussion, I thought It's a cell debris but media color showed me that it's contamination. It's cloudy. Thank you for Chidi Ahamuefula, Anand Laxmi and Stephen Sikkink for the responds.
Just a short comment on distinguishing between cell debris and contamination:
1. cell debris usually can be eliminated by washing the cells with buffer while contamination will remain.
2. the medium color with debris does not change (pink/salmon) while contamination results in acidification of the medium and the color turns yellow.
3. hazy or cloudy medium always means contamination by microorganisms.
you may try with your new primary to switch to new antibiotics: eg. from penicillin/streptomycin mix to gentamycin. We have good experience with plasmocin, it is effective against bacteria and in addition against mycoplasma.
Thank you for the comprehensive answer, Maria. After 3 days culture, Those points accordance with my primary cell culture.
Those images are almost certainly yeast colonies -- quite a common contaminant. The cells are bigger than bacteria and they often grow in clumps that look black by bright-field microscopy (as you have here), or shiny by phase contrast. The smallest colonies often look like chains or branching chains, as you can see in the second picture.
Yeast can sometimes be removed by fungicides, such as amphotericin, but if possible it is best to throw away the cultures and start again.
Getting rid of the culture is always the best way in case you can get new cells. You need, however, get rid also of the contamination origin. The dark picture is not very easy to interprete, but I feel that you had both baker`s yeast and a fungus in your culture, indicating contamination during handling of the culture.
The easiest way will be to observe the shapes and sizes if they are uneven then they are probably not contaminats however if there seem to be increase in size and change in shape they could be fungi or bacteria contamination. Thanks
Cells (adherent) are always always flat looking cells that you see in the image that you have uploaded. Debris will almost always be floating, irregular shape. Cells that look rounded and are still attached but seems to be rounded are dying cells (you don't want to see many of them in a nicely growing cell culture.
Those rounded clumps, my friend, are contamination probably yeast. Fungi will look like long threads.
One more thing. Depending on the cell culture such as microglia, cells sometime also grow detached from the surface and will look like clumps. Some of the transformed cells may sometime also grow as a clump but that will be towering up from an attachment point.
Best solution has been given by all who answered before me, throw these cultures away and start afresh and when you do, don't forget to add penn/strep (1%) solution to the growth media.
Good luck. Happy culturing!
Thank you very much Dorothy C. Bennet, Guenther Keil, Chidi Ahamuefula, Vincent Laize, Anuj Sharma for your comprehensive answers. I'll record all your suggestions in my note for evaluating my culture procedure.
Contaminations for sure! DO NOT attempt to recover the culture with antibiotics (unless the are rare and unique cells). You can never be sure that you got rid of the infection completely and can never trust the results you will get. Antibiotics can also damage the cells and affect their behaviour. As suggested above, just start with new cells and new solutions. The good news is that if that's the first time it's happened to you, it's probably just a technical error and not a deep routed source of infection in the equipment/surrounding.
When I evaluated my culture procedure, I found that the problem was in the waterbath. My labmate forgot to add some aquaquard or water disinfectant into waterbath. I think That's the answer. Thank you Nicolas for a good advice.
Dear, Gamze Can.
I didn't identify what kind of fungus is. I think It's just common fungus which you can find in the dirty water. Just need to keep clean your lab tools.
a situation like this happened to me too. According to the the attached image, do you think there is contamination or cell debris? I am sorry for the quality of the photo. ı took a picture by mobile phone.
What cells are those, can you take a pic of the cells in a mounted camera if possible
When cell culture get contaminated you will find many black or opaque large irregular masses often floating in the media while cell debris is not so opaque and large, fungus contamination occurs when sterilization of media is not proper.
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