For 96-square deepwell microtiter plates, culture volumes between 0.5 ml and 0.75 ml are typically used, preferably at 300 rpm on a shaker with a 50 mm amplitude (photo and video on the right). For 0.5 ml cultures, these shaking conditions lead to oxygen transfer rates (OTR) of 38 mmol O 2 l -1 h -1 (corresponding to kLa values of 200 h -1 ). Such an OTR - in its turn - corresponds to the high end achievable in Erlenmeyer shaker flasks (culture volume 10% of total flask volume, 300 rpm), and furthermore ensures mixing down to the bottom (no dead zones). The latter is especially relevant for large and heavy cells (such as yeasts and CHO- cells) that tend to precipitate in dead zones. Some groups use culture volumes of 0.6 ml instead of 0.5 ml, which still results in an acceptable degree of mixing.
25 mm shaker amplitude 300 rpm (full mixing up to 0.25 ml culture volume)
Hydrodynamics inside wells of a 96-square deepwell microplate
The mixing times under the above conditions are less than a second, as determined by adding a drop of acid to an alkaline solution (containing a pH indicator), and following the colour change with a high-speed camera (in the video at the right for a volume of 0.5 ml, resulting in a mixing time of 0.8 seconds).
50 mm shaker amplitude 225 rpm
OTR: n.d. 12* 7* 3* mmol O 2 l -1 h -1 * Duetz et al. (2000). Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Applied and Environmental Microbiology 66:2641-2646
OTR: n.d. 38* 24* 18* mmol l -1 h -
1 preferred condition
50 mm shaker amplitude 300 rpm (full mixing up to 0.5 ml culture volume)
Still from high-speed MP4 video below
Still from high-speed MP4 video below
Still from high-speed MP4 video at the right