When using a preculture with Ampicillin in my protein expression, I have to get rid of the preculture medium to avoid carrying over too much beta-lactamases that will destroy the ampicillin in the main culture. To do this I pellet the cells and resuspend.

Which methods of resuspension are best suited if you need the bacteria alive afterwards? I'm not sure how much force I can apply without harming the bacteria, and resuspending very gently takes a very, very long time.

Which methods can be used to resuspend bacteria alive, and which ones are the fastest?

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    $\begingroup$ I wonder how many cells are even killed in a pellet? It seems they always come back into log phase growth in a short time. $\endgroup$
    – shigeta
    Mar 4, 2013 at 13:38

2 Answers 2


Assuming that you are talking about E. coli: As long as you are resuspending the cells in a suitable liquid, e.g. fresh medium or buffer, then from my experience you don't have much to worry about - the cells are very robust. I've found that different strains and growth conditions give pellets with very different qualities - some will resuspend quite well with vortexing. If the pellet seems clumpy then trituration is something that I always found to work - pipette up and down until the pellet breaks up.

If you were going to do some kind of physiological measurement with the resuspended cells then it might require some checking, but if you only intend to regrow and induce expression I would say that giving the cells some time to recover in the new medium (check that absorbance is increasing) before inducing expression should be enough.

  • $\begingroup$ Using a pipette is what I'm doing now, though I usually cut off the tip a bit to reduce the force when pipetting. $\endgroup$ Mar 4, 2013 at 13:43

Pipetting up and down GENTLY, the slower you pipet and the thicker the tip the more gentle it is, or vortexing SLOWLY, high vortexing speeds result in shear stress on the wall, just like fast pipetting with small tips

Bacterias can take a lot, take more care with yeast and even more care with mammalian cells


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