how to solve the problem cell culture contamination? - (Jun/28/2006 )

Hi all,

I am doing stable transfection of my Chang's liver cell. They are adherent monolayer. After selection for 3 weeks, some of the dishes show signs of contamination by bacteria.
The medium (DMEM + FBS without P/S) turns a bit turbid. I can still see the cells at the bottom but there are bacteria floating.
I have renewed the medium, PBS and trypsin and washed the dishes using PBS for 3 times. And change medium. I have also tried to trypsinized the cells and centrifuge with 1000rpm for 10 mins to remove the bacteria, but it is no use.
As I am doing neomycin selection, I don't know if I should add P/S to the medium. What should I do to solve the problem? How can I get rid of the bacteria without killing the cells?
I don't want to do the selection from the beginning again.
Thanks!

If your medium doesn't contain any antibiotic, it will easily get contaminated: it's a very rich medium with whatever bacteria can need to grow. Moreover, your cells are incubated at 37 degrees and that's the best for the bacteria that usually contaminate your medium...
When I do G418 selection, I always use pen/strep as well. It doesn't hurt the cells if you use 1% pen/strep. And you will get rid of the contamination.
Sorry, but you cannot remove the bacteria centrifuging the cells. In my opinion, you should do the selection again. Sorry!

Always add the pen/Strep else u will get bacterial contamination.

As dnafactory suggested, do the selection again. Its better for ur experiments. If u get any data u might always b worried that it could b an artifact of contamination.