Hi All,
I have a cell slurry produced after infection with adenovirus (media + dead/broken mammalian cells + virus) and unfortunately, I believe that this slurry has a bacterial contamination. However, the virus purification SOP includes a 24hr spin at 20k rpm which I'm wondering if anyone has any anecdotal data (not hearsay) to show that that much time and centrifugal force has a bactericidal effect? This centrifugation should "clean" my prep regardless, but I was hoping to hear that the bacteria would be dead not just partitioned away. Additionally, the slurry I mentioned has been frozen at -80, and I was also wondering if freeze/thaws might help. I work with adenovirus a lot so I know it can withstand multiple freeze/thaws without too much loss of titer, so if I could freeze/thaw my slurries it might shred up the bacteria but maintain (some) of my adenovirus. Any thoughts?
Thanks so much for your help!
Of course high speed centrifugation can burst cells resulting in slurry formation. Frequently, it is a function of other factors especially pre-treatment with cell wall or membrane compromising agents. media components could also play a significant role
Dear Tiger
i'm not an expert of adenovirus, but if i well undersand its size is quite smaller (about 33mn http://www.wyatt.eu/fileadmin/applicationNotes/biopolymers/Multi_Angle_Light_Scattering-adenovirus.pdf) respect than bacteira.
Can you separe the broken cells from the virus and surnatant with low speed centrifugation applied prior ultracentrifugation?
Because in this case you can simply filter your cell surnatant containing the virus using 0,22uM filter. Theoretically the bacteria will be retained from the filter while the virus particle able to pass trough it.
good luck
Manuele
You could try ultra-sonication. Freezing thawing may not get rid of bacteria completely.
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