For 96-square half deepwell microtiter plates (CR1496c, specifically designed for cultivation purposes), culture volumes between 0.25 ml and 0.35 ml are typically used, preferably on a shaker with a 50 mm amplitude. The optimal shaking frequency depends on the culture volume. For 0.25 or 0.3 ml cultures, 225 or 250 rpm is generally used. Oxygen transfer rates (OTR) for this kind of microplate have not been measured yet. Extrapolation from the data obtained with 96-square deepwell plates (Duetz et al., 2000, Applied and Environmental Microbiology 66:2641-2646) leads to an estimated OTR at 250 ul at 225 rpm of 40 mmol O 2 l -1 h -1 (corresponding to kLa values of 200 h -1 ). Such an OTR corresponds to the high end achievable in Erlenmeyer shaker flasks (culture volume 10% of total flask volume, 300 rpm), To ensure full mixing down to the bottom (no dead zones), however, it may be advisable to use shaking frequencies of 250 rpm instead of 225 rpm. Even higher shaking frequencies (275 or 300 rpm) may be applied without the culture fluid touching upon the cover, on the condition that no higher culture volumes than 0.25 ml are used (see the bottom two video stills on this page. If only a shaker with a 25 mm amplitude is avaialble, shaking at 300 rpm (and a culture volume of 0.25 ml) is also reported to give good results (hydrodynamics and oxygen transfer rates comparable with 500 mm/225 rpm)
Hydrodynamics inside wells of a 96-square half deepwell microplate
96-square half deepwell plate CR1496c
Adviced (default) condtion 1: 25 mm shaker amplitude, 300 rpm (E. coli culture on NB, 20 times slowed down) 0.25 ml 0.3 ml 0.35 ml (full mixing up to 0.25 ml culture volume)
Adviced (default) condtion 2: 50 mm shaker amplitude, 225 rpm (E. coli culture on NB, 20 times slowed down) 0.25 ml 0.3 ml 0.35 ml (full mixing up to 0.30 ml culture volume)
Below: video database showing alternative shaking conditions (using E. coli cultures of 0.25, 0.3, and 0.35 ml, our advice: only apply any of the below conditions if you have a good reason to deviate from the shaking conditions shown above)
25 mm / 225 rpm
25 mm / 250 rpm
25 mm / 275 rpm
25 mm / 325 rpm
25 mm / 350 rpm
50 mm / 200 rpm
50 mm / 250 rpm
50 mm / 275 rpm
50 mm / 325 rpm