Have been finding these weird-looking objects in my culture dishes lately. There are several strange things here, but I am most concerned with the spore-like structure near the center of the field of view. It looks like filamentous branches with bulbs at the ends of the filaments. They are attached to the cells and don't come off with rinsing. The small black dots outside the cells are normal for this cell line, but I am also concerned about the larger, dark spherical objects. They are free-floating and come off with rinsing, but return quickly. I am wondering if perhaps they are a byproduct of cell stress being caused by whatever that filamentous structure is. My best guess is fungi? Any help is greatly appreciated.
Have you tested your cells to see if you have Mycoplasma contamination? They have kits for this.
Skylar King My understanding is that mycoplasma contamination would not be visible to the naked eye? Regardless, yes, I tested for mycoplasma and it was negative.
You can actually see mycoplasma. Glad it was negative though. How are your cells? Are they dying more frequently? Do you get this immediately after passaging? I notice if I do not change my medium every 2-3 days my medium starts looking funky.
After you see this do you notice your media changing colors to like a yellowish hue?
Well, the cells I'm working with grow very quickly and need to be passaged every other day. I haven't noticed more cell death than usual, but this is a very resilient epidermoid carcinoma line that is difficult to kill, so I'm not sure that is a good barometer. As for the media color, it does get yellowish, but that is due to normal acidification of the media over time. It isn't getting yellow faster than normal. I don't see those filamentous structures right after passaging, but I do see them once the cells are attached and starting to spread. And I see them regardless of the color of the media or the confluency of the plate.
Skylar King Also, how would you be able to see mycoplasma? Do you have any example pictures? They are smaller than the diffraction limit of light so I don't understand how they would be visible
Dear Will,
in the absence of contamination ( re check using Amphotericin B), it could be a possibility that the attached cells are undergoing differentiation. Due to the fact that post attachment filamentous nature is observed. Do a flowcytometry on these cells measuring the lineage markers for your cell line to understand any differences.Hope this helps you.
Hi,
So I have attached some pictures of mycoplasma, the bacteria surround the cell so you are seeing many bacterias aggregated and not a single bacteria, the cell culture does have to be quite contaminated before you would observe this. Also, I came upon this Fungal Myecilia image and I think you may be right that it could be fungus, since it does not go away with washing etc. Hope this helps.
Cell Culture Protocol 9: Testing Cells for Mycoplasma Contamination by Culture Isolation | Sigma-Aldrich
Figure 1 from Detection of Mycoplasma in cell cultures | Semantic Scholar (Nature protocols paper attached)
Fungus - UNC Lineberger
After treating cells for the second time with Amphotericin B (this time at double the normal concentration) the cells look drastically improved. I still found a few cells with those filamentous projections, but significantly less than before. I'm pretty convinced it was fungus after all. Hopefully after a couple more passages, the issue will fully resolve. Thank you all so much for your advice.
It may be a fungus, not Mycoplasma, because mycoplasma can't be seen!!
Regards.
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