Since last few weeks, I'm facing a problem with my resistant 231 cell line. After every third or fourth passage, the media turns a little turbid in 2-3 days, and I have to thaw a fresh vial. But it doesn't affect the growth rate or morphology of cells. And it seems to be fine under microscope. What should I do to avoid this?
Usually turbidity is associated with contamination. Check first the medium without cells for contamination 2-3 days with the same conditions as your cells. If the medium is not turbid, then try the oldest vial.
Sounds like bacterial contamination, although I would expect a change in medium color as well. I would suggest pouring an aliquot onto media used in bacteriology to check for bacterial of fungal growth. Be aware that not all bacteria grow on standard media; mycoplasma needs specific solid media combined with prolonged incubation. You may also check for the presence of bacteria in darkfield microscopy, applying 400x magnification. Again, this does not allow for detection of all kind of bacteria.
Stain your media kept in 37C for 2-3 days with Hoechst 33342. If the background is full of blue speckles, you have bacteria contamination.
One easy way,You can cultivate the 231 cell line without using antibiotics in the media. If it is contaminated, you will have no doubt in a few days. Antibiotics do not prevent contamination rather they control the growth of microorganisms.
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