I am facing a loss of viability in lymphocyte cells from human tonsils that I culture ex vivo. I have cultured dozens, but in the last months, small dots always appear in my media and the viability of tissue cells is very low (15-20%) when I usually get 60-80%.
The dots don't move and the medium is not turbid nor changing color. I tested for bacteria and it seems they are not bacteria. Could it be Mycoplasma contamination? Perhaps those black dots are cell debris from Mycoplasma-contaminated cells that cannot be seen by light microscopy?
I have attached photos. I also found that some lymphocytes are "budding"...
Thank you!
I'm not sure if you can discern mycoplasmas with phase contrast microscopy given how small they are. DAPI/Hoechst staining can help out here. Definitive test would be to use commercial kits, there are many kits but the one I used and that is popular is from Lonza: https://bioscience.lonza.com/lonza_bs/TR/en/Cell-analysis/p/000000000000186472/MycoAlert-Mycoplasma-Detection-Kit-%28100-Tests%29
It is super easy to use and gives quick results.
I would also agree with Justin...it is quite hard to see mycoplasmas with simple phase contrast mycroscopy. Mycoplasma are in the 300- to 800-nm range. These dimensions are close to the diffraction limit of visible light, where mamallian cells are around 10 000 nm..so at least 10-30 times bigger... you can use one of the popular kit out there... I could mention InvivoGen mycostrip...but I would be biased...
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