For 96-roundwell plates, culture volumes between 0.1 and 0.2 ml are used, preferably at 300 rpm on a shaker with a 50 mm amplitude. This shaking condition leads to oxygen-transfer rates of 20-40 mmol O2/L/h (corresponding to kLa values of 100-200 h-1). For a good mixing, we advice to use no more than 0.15 ml culture volume (or even less if higher oxygen-transfer rates are needed). Such low culture volumes make it difficult to take off the supernatant after centrifugation. A trick to make this easier is to add an equal volume (so e.g. 0.15 ml ul) of water or buffer prior to centrifugation. Please note that you have little room to manouvre with regard to the shaking conditions: a centrifugal force in the order of minimally 2.5 g (as with 50 mm/300 rpm) is needed to overcome the surface tension, and so to obtain full mixing down to the bottom of the culture, and prevent “ dead zones”
Hydrodynamics inside wells of a 96-roundwell microplate
50 mm shaker amplitude 300 rpm
Orbital shaking at an amplitude of 50 mm, 300 rpm Flashplayer movie (MP4-file) made with a high-speed camera and E. coli cultures growing on a mineral medium, 50 times slowed down
Still from MP4 video at the right