We've been trying to spin down a marine microalgae, Phaeocystis globosa. Other studies report centrifuging dense cultures at 100 x g for 10 min. We've been trying all sorts of speeds, durations, from 100 to 15,000 and end up with lousy pellets and a bunch of clumped-together cells floating in their seawater media. How can they resist being pelleted at high g? Why do these clumps form (cells not clumpy to start with). Shouldn't being clumpy make them more pelletable?
Dear David,
I have never worked with it but my suggestion is to try vortexing before centrifugation.
I don't think centrifugation at very high rpm as that of 15,000 is advisable.
Hope the paper mentioned below will be useful for your work.
Lipids, and their constituent fatty acids, of Phaeocystis sp. from the Arabian Gulf
R.H. AI-Hasan, A.M. Ali and S.S. Radwan,Marine Biology 105, 9-14 (1990)
Good luck!
Have you tried adding EDTA or TWEEN to the medium? EDTA inhibits calcium binding and TWEEN is a surfactant and may keep the cells from interacting. High speeds may disrupt the cells. Are the cells adhering to the centrifuge tube or just each other?
The best explanation I can come up with thus far is that because we're using a fixed-angle rotor, cells are getting crammed onto the wall of the centrifuge bottle but not sliding down the wall into a single pellet, thus forming sort of a streak of little pellets that then floats freely once the rotor stops. We have seen some evidence of this streak forming. We're using Pluronic acid in an attempt to improve our spins but haven't tried TWEEN yet. The media contains 100 micromolar EDTA (for trace metal buffering).
Also, I wonder if using swinging bucket would solve this 'wall-friction' problem.
I'll let you know if we figure it out.
Yes, try the swinging bucket if you have one. We've had similar issues with hemocyte separations, but we want them to clump and the streak is manageable. I was also thinking about a non-stick silicon treatment for the tubes, but that's not really the problem you're describing.
Hi there
I have similar problems in centrifugation of Ammonium oxidising bacteria. The cells float around even after spinning for 30 min. I do not want to increase the rpm above 10K since the pellet need to be preserved for further studies. Any help?
You probably need a surfactant in your buffer. TWEEN or something similar acts as one.
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