Am I Damaging My E. coli by Spinning at High Speeds?
Dear Aunt Yersinia,
A very annoying postdoc in our group keeps telling me off for spinning E.coli at 13K in a tabletop centrifuge. The postdoc claims that high speed damages cytoskeleton and this will reduce my transformation frequency. But I don’t believe her as the cells are cushioned by water during centrifugation. Can you tell me who is right?
Irene (PhD student)
I understand your sentiment – nobody wants to linger near a centrifuge for 10 minutes if she can spin cells in 1 minute. You are right – a bit. Nobody talks about the damage due to the movement through the liquid itself.
But if you jump out of a plane without a parachute, it’s not the fall that’ll kill you, but the meeting with planet Earth. A similar effect happens with centrifugation, and the higher the speed the harder the fall. Eventually the bacteria will meet the bottom of the tube and each other at the bottom with a considerable impact. Compacting under the centrifugal force will break the outer structures, for example flagellae and will damage the internal structures such as cytoskeleton and even DNA.
Gram positive bacteria are less sensitive to this because of thicker cell walls. However, gram-negative bacteria, including E. coli, will suffer. So if somebody studies bacterial cytoskeleton, motility or biofilm formation, they should forget about spinning at a maximum speed.
Despite, or maybe because of, being annoying – meticulous people are often grating – the postdoc is right. E. coli is susceptible to “over centrifuging”. Competent bacteria with their membranes treated in various ways to enhance their DNA uptake, even more so.
So what should you do? 3,000 RPM for 5 minutes mostly does the job. You do not need to exceed 4,000 RPM on a table-top centrifuge or any other to spin E.coli. Yeast are much larger and 3 minutes at 2,500 RPM will be enough to sediment > 95% of cells, although the exact result depends on your centrifuge (remember RPM does not equal RCF), so do try this at your own lab.
As for the boredom of waiting for the cells to spin, I recommend using this time to tidy your bench or a communal sink and you won’t notice how 10 minutes pass.
Peterson B.W. et al.(2012) Bacterial Cell Surface Damage Due to Centrifugal Compaction Appl. Environ. Microbiol. 78:120–5.
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Almost exactly the information I was looking for, but why did you put an RPM speed instead of RCF? You even reference they’re not the same thing, and the RPM in this article is not a useful number, so why not just tell us the actual RCF we should be using?