I did primary RPE cells extraction from the fresh pig eyes, but after 3 days of seeding cells the medium (miller medium including 10% pig serum and 2% penicillul/streptomycin) is looking cloudy( high number of bacteria). I am sure aboutn sterillization of Miller medium. The pig serum was negative control has bacterial contamination after two night incuzating in the incubator but when I incubate a plateas negtative control of miller medium incuudedl 10% of pig serum and 2% of pen/strs i donot see any contamination. Does anybody has the solution for this problem?
Hi there,
You should consider 0.22 um filtering your serum before use. It sounds like it has traces of bacteria that grow in the absence of PenStrep.
If possible I would also suggest 1 or 2 rounds of washing (via centrifuge) of your primary cells before you plate them. It is also OK to culture your cells and wash then replenish media at the earliest signs of bacterial contamination (before the media is cloudy). If you choose to do this I suggest you view your cells through a microscope multiple times a day as bacteria can become rampant in a matter of hours.
Thanks a lot for your reply.
I Think the washing cells before plating can help but with PBS or medium? how many rpm and how long Its needed?
once I filtered with 0.4 pore size the medium which already included antibiotic medium and this also showed containation after 3days . i never tried to in Rede the antibiotic concentration more than 2%. can it help?
Pbs or media, it doesn't matter so much for that period of time. I've always opted for media.
Again, speed will depend on the cell type. Having not worked with your cells I would guess that 200 g for 10 min.
This still showed contamination? Try 0.22 um and clean your incubator and hood thoroughly.
You can run higher concentrations of antibiotics in the short term. But I would not recommend past a few days.
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