I have grown 22Rv1 cells for virus transfection. After i added the virus the cells were clean but when i added puromycin for selection of stable cells then after 48 hours these black thread like structure appeared. I have performed this experiment twice but always i get the same black thread like structure in my culture.
Normally for culturing 22Rv1 cells i use RPMI+10% FBS+ 1% penicillin/Streptomycin. And for transfection i used RPMI+ 10% FBS and 1ug/ml puromycin for selection of stable cells.
Hello Sangeen Khan
I am afraid that these filamentous thread-like structures appear to be fungal hyphae/mycelium, indicative of fungal contamination. I understand that you used antibiotics for preventing any bacterial contamination and also added Puromycin for selecting your stable transformants, but fungal spores remain unaffected with this treatment. You need to ensure that your media, transfection solutions, and reagents are all filter sterilized. Fumigate your CO2 incubator and cell culture facility to avoid further contamination. Also, wear gloves and make use of 70% alcohol to wipe hands. Fungal contamination can easily be transmitted from hands and other fomites. When it comes to eliminating/avoiding any fungal contamination, people make use of cycloheximide. But in cell culture, the use of cycloheximide will inhibit protein synthesis in your cell line. Therefore, you may supplement your media with Caspofungin or Amphotericin B, which are a broad-range antifungals that will eliminate any contamination, be it fungal or yeast.
Hope this helps you :) Good luck!
I strongly agree with Jatin Chadha
Additionally from your experiment point of view, I would like to add that in general for transfection we use Opti-MEM (serum-free or reduced) media, not the regular growth media. If not Opti-MEM then go for the serum-free RPMI media for transfection.
Best!
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