preventing infection of cell cultures - (Jul/04/2004 )

Hi, I have been conducting research on neuroglial cells for about a year, but my research for the past year has been constantly beleaguered by infection. The occurrence of infection has been rather sporadic, and since I religiously follow the protocol and do not vary much in the way I do things, I think the protocol that I am currently following has left out critical steps to preventing infection. Does any one have any suggestions as to what is important in preventing infection?
I also have one specifc question about handling the media. Right now, I do not filter the media nor the antibiotic/antimycotic. The media is kept in its original container, and I simply add a bovine serum and the antibiotic/antimycotic to it. One batch of media will probably last me about a month. I know that you are using powdered forms of media, but does that apply to a working solution of media and the antibiotic/antimycotic, too?

In our lab, contamination by microbes is rare. I think the very important thing is aseptic techniques and good laboratory practice. Since I was trained as a surgical doctor and know the consequence if I fail to follow aseptic techniques. I don't know if your problem is related to this, but I do observe many PhDs don't have a concept of aseptic techniques.

In our lab we use cell culture technique a lot and don't come accross too many contaminations. We use liquid media in bottles and other reagents (Antibiotics, serum etc...) that should be opened only in the fume hood. I find that a month is usually the maximum that I want to keep a bottle for. If I don't think i'll use it all, I tend to make half a bottle and put it in an empty autoclaved bottle.
Our medias usually contain DMEM or equivalent, Serum, antibiotics (Penicillin, Streptomycin) and other nutrients such as Glutamine etc depending on the cell type.
Filtering the media might be a good idea if you have a doubt about the fact that some of your reagents are not sterile.

Hope that helps
Yasmine.