Hi Mahsa Khayat-Khoei

How long have you been culturing the cells? Some cell lines produce more waste than others, so this could be the reason.

Monitor your cultures for the next few days - if you do have a bacterial contamination, the media will turn cloudy and you'll be able to see millions of bacterial cells.

Do you have much cell culture or microscopy experience? It appears that your first two photos are on the wrong focal plane..

Wipe the underside of the plate and see if the dots and structures disappear or rearrange.

Thank you for your help.

@Ron Hrstka: I know the plane is wrong but I could see these black dots and the round structures on a different focal plane! So I took the first two pictures on a different plane on purpose. And the last picture is of the iPSCs to show how they look. And the black dots are not everywhere, and the media is not cloudy.

So I guess it makes sense that they are just debris. Maybe I left the enzyme on the cells longer than I was supposed to when passaging a few days ago.

I will keep monitoring the cells. It's passage 18 now so it's been a while that I have been passaging and maintaining this line and never had a similar issue.

Thank you again for your help,

Mahsa

There is a possibility of contamination but this looks more like dust particles and a stain form medium (bubble) on the underside of your plate. To test for contamination put some of the medium in a clean plate and incubate it for a week. If nothing grows you are safe. If it does clean everything, preform contracon of your incubator and start again. You know how it works.

Thanks @Mateo Glumac. Good idea! I will try that. I am very hopeful that they are just debris and not contamination...🤞

I wouldn't worry about it, I have the same structures when I culture them even changing the medium everyday and after doing bacterial contamination tests they were negative. I agree with Jack Hopkins