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Weird contaminant in culture? pics included - unknown contaminant (Oct/28/2008 )

Can anyone help?
We have had an apparent infection in several of our (siRNA treated prostate cancer) cell cultures. It's not like the usual bacterial infection (highly motile/media changes colour), its sporadically distributed small black dots which over 48h grow into larger colonies or chain-like colonies that frequently detach from dish and float in the media. The dots dont move, as far as I can tell, and I have attached bright-field images taken using the 10x and 40x objective.
I have had them growing for 7 days now and they dont seem to multiply exponentially like the bacteria we're used to, there are still relatively low numbers of them in the dishes.
They are growing in Hams F12 medium at the minute.
Does anyone know what this is?


Any Gram stain or agar culture colony observations?


Any chance of this being cellular debris?


This really looks like cellular debris but it's impossible to say for sure from a picture. Any chance that the siRNA is killing the cells? Do you see this in the control plate? How about your stock plates? You can take some of the media from your cells before the 48hrs, put it into a dish without cells and see if you get anything. If there is no change over the next couple days then you don't have any growth contamination and this is just debris. If you do see changes in the media I would think you've got a mold or fungal contamination.


I knew I would leave vital info out! Sorry, the pics are from a well that contained just medium+1.5ul transfection agent (siPORT neoFX), no cells at all. I often wondered if it was just cell debris due to it not moving so I did this test plate and 24h later I had a few dozen dots, at 48h there were hundreds. It didnt really grow up too much over the next 5 days.
We dont have any microbiology expertise at out lab although it would be nice to send it off for a proper diagnosis, so no Gram staining etc has been done.
Thanks for the replies so far!


It looks like clusters of detached cells to me, these would increase over time. siRNA/transfections quite commonly affect cellular attachment properties so I would guess you are having this problem.

It really doesn't look like either yeast, other fungal, or bacterial contamination.


Anyway, i would suggest you make tests with media alone, and with media plus transfection reagent. Maybe your media of the transfection reagent are contaminated.

Either way, you should get rid of these things. You don't want to carry contaminations over!


QUOTE (Paul_R @ Oct 29 2008, 05:48 AM)
Sorry, the pics are from a well that contained just medium+1.5ul transfection agent (siPORT neoFX), no cells at all.

Read what he said - it's just media + reagent..
It does seem very weird for a bacteria though. and the second pic just looks like detached cells, like the ones that float off with over confluency.
Check media alone, and the transfection reagent alone - perhaps one alone is contaminated, possibly even with cells.

-Aaron I-