Each vials that i thaw and grow becomes contaminated after sub culturing twice. Have used newly prepared medium, still experiencing the same situation. The cells are very well attached but contamination always occur when they are nearly 70% confluent.
Hi,
pls add the picture of contamination if you have
follow the Aseptic technique, Antibiotics, filtration (
Hi Sreedhar,
Yes i do follow all the aseptic techniques you mentioned above except filtration of media. I prepare my media in 50ml tubes prior to use and seal them. I did check if my media is contaminated by incubating the media in a dish and observed for 72 hours. It was clear.
Sound like your cell stocks are contaminated if all else is ok and everytime you get a new vial there is contamination. The problem with cell culture is that most people use antibiotics (Pen/Strep) which give a false level of hope, thus a low level of contamination under normal circumstances can take time to surface due to the presence of Pen/strep but also frequent passaging. Either seed a small volume of a fresh vial in to LB broth and also streak on to LB agar (no antibiotics): leave for 24+ hrs: do you get contamination: examine broth under microscope or visually. You may be able to examine the vial contents under a microscope and look for bacterial infection.
bw
Ian
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