Hello everyone. I am culturing hIPSC in mTeSR-1 medium. I found some small round white particles persistently attached to cell colonies, and some actively moved under the 40X objective. Also, black dots around the cell colonies move. I tried adding 1% penicillin-streptomycin (P/S) but it did not work well. Then I combined P/S with Primocin and Plasmocin for 5 days, but only black dots are reduced, white ones still exist in my culture. I could not find similar things when checking the medium. I also tried EtOH 70% disinfection followed by UV for the clean bench and cell culture room, but I could not eliminate these contaminants.
Is it bacterial or fungal contamination? Can you suggest any method to rescue my cells?
Thank you very much.
Hi,
to be honest, I am not completely sure you do have contamination. How does the medium look - cloudy in any way?
The white spots look like dead cells to me and when these dead cells disintegrate, they leave those tiny black dots. So it is also a question of whether the black dots are moving and forming chains - that would indicate bacteria. When you split/passage your iPSC, do you still see the same amount of black dots?
Furthermore, if you have the feeling your cells are not looking as before or are not growing the best way, are you testing for mycoplasma? They would not be visible under a standard tissue culture microscope so other tests like PCR are necessary.
Good luck with your culture!
Selene
I agree with Selene. Since the white spots are not out in the media and only over the cells, I don't think it's any kind of contamination. The movement you see is probably due to vibration in the room.
Cells round like this and become refractory when they enter mitosis. This likely is what you are seeing.
The small round white particles and the black dots visible in your cell culture are likely indicative of contamination. Given that the particles are persistently attached to the cell colonies and move actively, it suggests the presence of a biological contaminant, possibly bacteria or yeast.
Identifying the Contamination
Based on the images and your description, here are some possibilities:
Suggested Steps to Address Contamination
Long-Term Solutions
- Backup Cell Lines: Always maintain backup stocks of your cell lines in liquid nitrogen to avoid total loss in case of contamination.
- Supplier Assurance: Ensure that all cell culture materials are sourced from reputable suppliers and are quality-checked.
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