Media turns Turbid after 3 days of culture - (Feb/01/2008 )
Hello
I have been having the same problem for more than 8 months now. Now I am not sure if this is contamination or just normal!
I am running a bioreactor with 6 tissue samples. I used to seed 1million cells/ml. Thus a total of 6 million cells in the whole reactor. When I tried to seed 10 million cells/ml, thus 60 million cells in the reactor, I started noticing that the media is turning turbid after the 3rd day of culture. I change my media 3 times a week (MWF) and every time I change it I notice that it turned turbid. The color is still normal red but you cannot see through the media any more. Also at the bottom of the bottle there is a thin white layer.
First I thought I might have some kind of fungal contamination. SO I sterilized all the components of my bioreactor with hot base and changed all my tubing. I started culturing new cells and I repeated the experiment over and over again and I always get the same result! The media turns cloudy. When I look under the microscope I just see debris. I cannot tell if there is any fungal contamination. Please help me. How can I figure it out. Is it normal at high cell densities for the media to turn turbid?
One last thing, I do not get the turbidity when my cells are cultured in T-75. So I know that the cells are not contaminated at all.
Thank you
Would turning the spinner/agitator speed down help? Too much shear force will rupture some cells.
Thank you for your reply. My flow rate is extremely low. However, I was suspecting since my seeding density is too high I might have mass transport problems through the tissue and into the cells. SO some cells might be dying or lysing. But would this cause turbidity?
Perhaps then, the media is turbid becasue your cell density is so high.
Thank you again. So high cell density does cause media to be turbid? Did you experience this in other cases?
That's what I think too.
Just take bacterias in LB. The media becomes turbid, but as soon as you spin down the cells, the media becomes clear again. So yes, a high density will cause the media to be turbid.
Just take bacterias in LB. The media becomes turbid, but as soon as you spin down the cells, the media becomes clear again. So yes, a high density will cause the media to be turbid.
It is good to know that I don't necessarily have contamination. I have repeated this experiment several times within the past year. It takes me at least 2 months to get everything ready for the setup. I always get the turbidity and conclude contamination. I am trying to graduate with a PHD degree and I told my adviser that if I had to go through this one more time I am going to quit. It is so challenging and unrewarding to work for a full year and have NOTHING. With a civil engineering master degree I was used to work hard and get results. BioEngineering is so different.
I thank you for all the input you gave me. I guess getting contamination every single time I set up the experiment does not make sense. I believe now that your explanations are more reliable. Maybe it is my high seeding density after all. Thank you all for the help and if you have any additional comments that might help further I would appreciate it.