Effectiveness of orbital shaking for the aeration of suspended bacterial cultures in square-deepwell microtiter plates.

Growth of heterogeneous culture collections in microtiter plates is advantageous for logistic reasons and also in enabling significant savings in medium costs, labor input and use of equipment during large screening projects. The main hurdles to overcome for aerobic microbial strains are the prevention of cross-contamination and excessive evaporation while assuring sufficient aeration rates. For this purpose we developed a sandwich spongy silicone/cotton wool cover to close the wells of square-deepwell microtiter plates. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and were shown to be threefold higher during orbital shaking at a shaking diameter of 5cm at 300rpm (24mmolO(2)l(-1)h(-1) at a culture volume of 0.75ml) in comparison to a shaking diameter of 2.5cm. Photographic analysis showed a clear influence of the shaking diameter on the hydrodynamic behavior in the wells; during shaking at a 2.5cm amplitude, out-of-phase conditions occurred resulting in poor vertical mixing, while a 5cm shaking amplitude led to an optimal surface to volume ratio and a turbulent flow.