I've ran a time series imaging experiment for CHO cells where I replenish the media and image a 24-well plate every 24 hours. These cells are under Puromycin treatment and some are induced to be resistant. In wells that have very few cells, I'm noticing small black dots that wiggle around and move near the center of the well. I have a video and some images of the objects. I'm worried that it's Achromobacter. Has anyone dealt with this issue before? Maybe during puromycin treatment?
I had a similar experience with my S2 (Drosophila) cells. I was also using puromycin and thought the black dots that I was seeing may have been some type of cell debris. The viability of my cells also began declining the longer they were in culture with these black dots. I decided it was contamination after I attempted a cell lysis with RIPA buffer, but I was still collecting a cell pellet after centrifugation. Whatever type of contaminant this was, it apparently could not be lysed by RIPA. I had to discard several plates, clean all of our hoods and incubators, and start over. Based on your images, this might be a similar problem.
Ok, thanks for the advice.
RIPA is typically used to lyse mammalian cells and apparently doesn't work for bacteria. Did you try any other method to come to your conclusion? Was the media cloudy? My media is pretty clear.
My stock media was clear, so it wasn't contaminated. But I could see some unusual cloudiness in my plated cultures. It looked kind of brownish. But I didn't try anything else to confirm the contamination. I just knew that RIPA can very effectively lyse my S2 cells, so when I saw that I was still getting a cell pellet I knew there had to be something other than S2 cells in the cultures.
Most likely cell debris given size (some nearly 10um long) and puromycin treatment. Note that cell debris itself can be toxic for cell culture and therefore should be cleared, otherwise you may see adverse growth effects due simply to the presence of cell-free DNA, proteases, etc. (which also, by the way, can make your media slightly turbid, although nowhere near that of a true microbial contamination). A cell pellet after RIPA lysis could simply be free protein/DNA complexes from dead cells (in effect, they were lysed prior to RIPA treatment) and does not necessarily indicate a true microbial contamination. However, difficult to be certain by imaging alone. Best would be to sample media, spin down gently to remove floating cells, and then grow the "contaminated" media to see if the black dots proliferate. Failure to proliferate is consistent with cell debris.
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