washing and centrifugation of microorganism without losing concentratio - loss of microorganism via repeated washing? (May/23/2006 )

Hello everyone,

we are trying to achieve a higher cell concentration than the overnight culture yields by washing and centrifuging our organism and then resuspending it in fresh growth broth for another round of growth yield.
Using that second round of growth culture we centrifuge it at 4000 g’s (is that too slow?), decant the supernatant and wash in deionized water. With the first centrifugation step we see a nice pellet, though with the second centrifugation step the nice pellet seems to have disappear unless the “dirt” from the media got washed out though the washing step and we just have to assume that there are cells in a pellet somewhere in the tube?
I am just concerned that we are losing microorganism via the washing process. We are trying to achieve 10^11-10^12 cells/ml.
I have also noticed that some books recommend 4000 g’s other’s 8000 g’s …what’s the recommended speed for centrifugation of growth culture to receive a nice pellet loaded with microorganism.
Also, can one centrifuge the organism at a lower speed but for a longer time? If the centrifuge can’t go beyond 4000 g’s? I wouldn’t see why not.
Thanks for your input.

Bacteria can withstand centrifugation speed far in excess 8000 rpm. So no worry here.
Centrifugation for a longer time at lower speed is an option, but in my experience the pellet is usually more loose than with a higher speed when possible.

As for your strategy to increase your bacterial concentration:
-Choose your medium to get better yield. It depends what species you are working with. In a previous thread I think Terrific broth was recommended with E. coli.
-no need to wash with deionized water, you are harming your bacteria, using the growth medium is way better! And there is no such thing as significant dirt in your pellet. It's just probably to loose when you wash.
-Grow your bacteria in bigger vessels and concentrate by centrifugation (1L that reach 10E9 bacteria/ml is 1 ml at 10E11/ml)