Cell Culture Decontamination

ATCC has had some success using the following combination of antibiotics to eliminate bacterial contamination of a valuable cell line.

Prepare complete culture medium containing final concentrations (1x) of: 250 U/mL penicillin, 250 µg/mL streptomycin, 250 µg/mL neomycin and 2.5 U/mL bacitracin. Gentamicin sulfate is an antibiotic and is used at 50 to 100 µg/ml culture medium. The antimycotic amphotericin B is used at 2.5 µg /ml culture medium

Grow the cells for 2 weeks in media containing this antibiotic combination. Be aware that the cells may not tolerate well the increased concentrations of these antibiotics.

After treating the cell culture, continue culturing in antibiotic/antimycotic free medium and retest to make sure that the culture is clean.

Continual observation and periodic testing is necessary to make sure that the contaminant does not reappear. Since many antibiotics may be toxic to cells, a selected population that no longer exhibits qualities of the parental line may result. It may be necessary to examine the cured culture to assure that it retains properties sufficiently similar to the original line. This antibiotic mix can be used to remove bacteria from primary tissue when establishing new cell lines. It is used at ATCC to establish primary fibroblast cells. It is also effective in minimizing microbial contamination during FACS procedure. Be aware that this combination does not include an antifungal therefore, depending on the purpose, one may need to add amphotericin B.