I work on the U87 glioblastoma cell line, I culture the cells in DMEM medium. I put my cells in culture. I checked them for a week and I didn't find any problem, but after some days all the cell were contaminated. I did all the tests, changed the material, protocol, people handling it, lab, but this keep happening. We culture the cells, they are good for a few days and passages but they keep contaminating!!! I dont know what else to do
Hello Maria Eduarda Marques
You need to strictly follow sterile conditions while you are working in Tissue Culture Laboratory.
In brief, all the tissue culture wares have to be autoclaved before use; the cell culture media has to be filter sterilized using 0.2um filter; the uv lamp has to be switched on for 15-20 mins before you start working in the biosafety cabinet; lab apron, hand gloves and face mask have to be worn while working in the laminar hood; one person should work in the hood at any given time; avoid crowding the Tissue Culture Laboratory.
Since you have been facing the problem of contamination repeatedly, I would suggest you fumigate the Tissue Culture Laboratory. Maybe this could help solve your problem. I too faced a similar problem (repeated contamination), but after fumigating the laboratory, I can now work efficiently.
If you are interested in performing fumigation, there are some points you need to bear in mind whenever you fumigate the Tissue Culture Laboratory.
1. Ensure that AHU and AC systems are in OFF position.
2. Maintain the temperature of the laboratory at 21-25 degree C and the humidity at 70-80%.
3. Add 25-30 mL of formaldehyde solution (37%) to 10-15g KMNO4 per one cubic meter of space in a glass container that is quite sufficient in size so that the material does not expel out of the container. When mixing with potassium permanganate for fumigation, always add the formalin to potassium permanganate and not the other way.
4. In a similar manner, use 5g of potassium permanganate and 10-15ml of formaldehyde solution for the airlock.
5. Use multiple containers and distribute them throughout the laboratory.
6. Ensure that the entry points are sealed, and the containers may be left overnight in the laboratory.
The next day, you may switch on the AHU system and give an exposure time of at least 4 hours before entering the laboratory. You may neutralize the area by placing ammonia solution in the center of the laboratory and leave it for 3 hours to neutralize the formalin vapor.
To avoid any injury during the process of fumigation you need to take certain precautions.
Always wear mask and goggles while you perform fumigation. Beware of splashing as the reagents react. The reaction generates heat. Avoid inhalation of the fumes. Also, ensure that the entry doors of the laboratory are sealed after performing fumigation.
Good Luck!
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