I found some dot particles after reviving. They have not washed away after 2 to 3 passages. The media colour is not changing even though the particles are huge. The particles are not moving. I attached the picture, please give your suggestions to understand what they could be.
What is the concentration of antibiotic that you are using? Are the particles increasing over time? Whether they are attached or in suspension? The particles in the picture are not very clear. Can you please provide a picture with higher magnification?
These look like contaminants which may be growing slowly because of the effect of antibiotic in the media.
Thank you for the responding. I am using pen-strep(P4333 SIGMA) i.e.,100units of penicillin&100ug of streptomycin per ml finally. These particles doesn't have specific morphology. But these are increasing when the cell confluency is more. Its like less cells less particles. I attached one more image when confluecy is high. Please suggest possible solutions.
Next time you subculture your cells, grow some cells in media without any antibiotic. Check for contamination and discard the whole batch of cells that you are maintaining and start with a fresh culture.
Mr. Sonowal, I did revived new vial, and subcultured in antibiotic media for 4days with no contamination. Now I am conducting my experiments with them. Thank you for your valuable suggestion.
its good culture practice to gram stain and test for mycoplasma every 1-2 months in culture. both are relatively easy and cheap to do as well.
Yes, it was the debris formed from the Cells(esp. from dead cells formed when they were grown in two layers). Need to change media in the second day of spliting and split cells when they are below 80% confluence.
I am facing the same problem as well. There seems to be black dots that are attaching/sticking to the growing cells and are not going away even after washing with PBS or changing media.Please advise. Repeated use of fresh vials stored in LN is also not helping the situation.
Hi Santosh,
Could you please upload images of your culture which gives more information to give most probable answer.
Facing similar problem. Debris in the background, which i think is from necrotic cells. These cells were growing fine initially, clean background and morphology, but suddenly such a change, over 1 - 2 passage. Media used to earlier be clean, but now slightly murky i think due to these particles.
What did you find, is it contamination? that is triggering cell death?
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