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Contamination? - (Aug/17/2009 )

I am culturing small cell lung cancer cell lines, and recently I have run into a problem, or at least a question. "Dark bodies" have begun to appear in my cultures of H69, H510, HAR cell lines. The number of "dark bodies" increases over time, so they being produced in the culture. I believe they are apoptotic cells, but I'm not sure. It is not uncommon to see some dead cells in these cultures, but they usually are more translucent.

--- I had this problem before, I made new medium, and it went away. This time I made new medium, and it did not help. So, I made another batch of medium and filter-sterilized it (0.2 um) after adding serum (usually I filter and then add serum). This also did not help.

-> This would seem to rule out fungi or most bacteria, leaving mycoplasma and viruses as possible contaminants. However, I cannot figure out how they might be getting into the culture.

--- I have autoclaved the incubator racks and sprayed the incubator interior with 70% ethanol.

--- I have started several cultures from frozen stocks with new medium. I have used stocks from different batches that have produced good cultures in the past.

The problem persists, if it is a problem. While this is not how my cultures usually look, I was wondering if anyone knew if it was normal for this to periodically happen in these cell lines.

Any ideas, suggestions, etc. are greatly appreciated.

A couple of pics...
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Maybe you should take a very small amount of what ever it is and seed it onto a seperate tissue culture flask with no cells, just tissue culture media. If it grows then you know you have something in there that shouldn't be in there.
Sometimes when my cells (renal proximal tubule cells) aren't particularly happy they don't adhere and form these dense clusters that vaguely resemble what you are seeing, but when you change height of the microscope you can see that they are just cells. Maybe have a look and try adjusting your microscope height? They could be just cells that did not adhere and have clustered together?


I appreciate your comment.

Unfortunately, I forgot to mention that these cell lines grow in suspension cultures, so clustering is normal. The dark bits are found in the clusters, but mostly on the flask surface.

I have tried culturing for the contaminant in several ways, including the way you mentioned and also on LB. Nothing grew in any case, so it seems that if the problem is biological, it is probably a parasite (maybe intracellular).