Hello everybody, i hope somebody can help me with my cells. Since 2 weeks they started to change morphologically, as you can see in the two pictures with the smaller magnification (they started to build a vessel like formation combined with some really dark round structures (this is no pigment!)). I really dont know what the problem is but i have had this now every time after RPE isolation. (I tried new media, we cleaned the incubator etc. but still it looks kind of contaminated). In the third picture (the one with the larger magnification) you can see that there are a lot of small black dots which even moved (bacteria ?). So my overall question here is, if those cells look contaminated to you ? And if yes what can i do to prevent it in the future even though i already threw away all the old media, and openend new bottles for everything, cleaned the incubator etc. ??? Thanks a lot for your help, Peter
The morphology, structure, and plenty of small spheres resemble fungal contamination. Sterilize the incubator with a high temperature and pour a clean reliable water in incubator water reservoir and add some copper sulfate to the water to prevent fungal contamination.
Could the black dots be differentiated cells producing pigments?
On the third pic, many cells look dead/fragmented.
Otherwise, moving particles usually are not good. Are you using antibiotics? Which? To get more clues, you could streak a little medium on agar plates w/o antibiotics.
During the isolation of primary cells, it's easy to introduce contaminants, so you might want to add antimicrobials at least for a little while.
If you really suspect systemic contamination and your incubator is made from stainless steel (no copper), you might put a bowl with some acetic acid inside and heat it over the weekend to about 60°C. Let it cool down before opening it again and have good ventilation. Kills all microbes in my experience and is a little less nasty than fumigation with formaldehyde.
I think it is fungal contamination. Clean everything possible. Fungal contamination is difficult to get rid of but it is a common problem for researchers. Change the media water everything. Use potassium dichromate in incubator water. Clean the whole hood ,room and specially the incubator. Perform contracon. I am quite hopeful the problem will be solved.
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