I am culturing primary human diabetic dermal fibroblasts using growth media during the culturing process I have encountered contamination so started to use antibiotic antimycotic. Now as anti anti concentration is increased the contamination is not there but the cells are dividing so slow it is taking almost 2 weeks now yet the plates are not confluent. Kindly provide me with suggestions.
Hello Fathima Beegum
While working in a tissue culture facility, one must maintain sterility. If sterile conditions are strictly followed, there would be no need to add antibiotics in culture media. Ideally, mammalian cells should be cultured without antibiotics/antimycotics.
If at all, you would want to use antibiotics in cell culture, you should use it at an optimal concentration. Between 0.5 and 1 mL of Penicillin-Streptomycin solution is added to 100 mL of cell culture media for a final concentration of 50 to 100 I.U./mL penicillin and 50 to 100 μg/mL streptomycin. But please note that it should not be used as a replacement of sterile practice.
Antibiotics and antimycotics can be toxic to certain cell lines. Antibiotics deeply influence cell metabolism. Therefore, it would be essential that you first perform a dose-response test to determine the level at which cell toxicity begins.
I would suggest that you try to keep the cells away from microorganism contamination which may be accomplished with proper knowledge of good laboratory practice. Following all the guidelines towards maintaining a sterile condition will make these compounds unnecessary in cell culture. In short, maintaining a sterile work area, sterile reagents and media, and good personal hygiene would help to create a sterile environment in cell culture.
Best.
When I isolate tissue for primary cell culture I "wash" it by transferring it throuh 3 sets of dishes of basal medium with pen-strep and gentamycin for 10 minutes each. I use fresh, sterile forceps for each transfer. The first 24 hours in vitro is done with pen-strep, then changed to antibiotic- free Medium
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining