Hydrodynamics/OTR recommendations and background

In order to keep your suspended culture in the exponential, non-oxygen-limited, growth phase as long as possible (and to prevent settling of cells), it is essential to perform cultivations with the right culture volumes and shaker settings. This section of the website can be used as a reference for the recommended culture volumes mentioned on all product pages of microplates. With these recommendations, one is able to achieve an OTR in the range of 30-40 mmol O2 l-1 h-1, which is similar to OTRs typically achieved in stirred tank reactors.

The hydrodynamics of a culture inside the wells of an orbitally shaken microplate are determined by: 

  • the shape of the well: square wells result in a higher turbulence than round wells. This effect is comparable to - but milder then - the effect of baffles inside an Erlenmeyer flask. The extra turbulence caused by the square shape is mild for 96-well plates, and somewhat stronger for 24 well plates, especially at higher shaking frequencies. 
  • the diameter of the wells 
  • the culture volume
  • the shaking frequency 
  • the G-force excerted by the orbital shaker movement. The G-force is proportional to the shaking amplitude, and the square of the shaking frequency: G- force= 5.6 x 10 -7  x shaking amplitude x rpm 2  (with the shaking amplitude expressed in mm). The G-force in its turn determines: o the angle of the culture fluid with the horizontal plane. As a rule of thumb (to achieve a full mixing), this angle should be in the order of 50 degrees for 24-well plates (corresponding to a G-force of 1.2, that can be established by shaking at 50 mm/225 rpm, or 25mm/300rpm), and 70 degrees for 96 deepwell plates (corresponding to a G-force of 2.5, that can be established by shaking at 50 mm/300rpm)

In practice
Knowing the G-force (calculated from the shaking frequency and the shaking amplitude), and so the angle of the culture fluid with the horizontal plane makes it possible to predict to what extent the culture is fully mixed (down to the bottom of the well) for a certain culture volume. However, also other factors may have an impact, most notably the surface tension (especially relevant for wells with a diameter below 8 mm, such as the wells in 96-roundwell plate), and the degree to what extent the culture fluid is capable of fully following the movement of the shaker (especially relevant for square wells in 24 well plate, shaking at smal amplitude, and a high frequency).

Therefore we have also made videos of shaking wells using a high-speed camera for a range of types of microplates, with different culture volumes and a range of combinations of shaking amplitudes and shaking frequencies. These videos allow you to judge for yourself if a specific shaking condition is suitable for your application:

Furthermore, we have compiled lists of oxygen-transfer rates from literature data. When comparing these measure maximal oxygen-transfer rates with the videos of shaking wells under the same shaking conditions, the following patterns/rules of thumb become apparent:

  • If the cultures are not mixed down to the bottom of the wells, the OTR is below 30 mmol O2 l-1 h-1
  • If the cultures are mixed down-to-the-bottom (just, but no further), the OTR is in the range of 30-40 mmol O2 l-1 h-1
  • If the cultures are mixed to the center of the wells, the OTR is 50 mmol O2 l-1 h-1 or more


Contact us

The best way to contact us is to use the form below. However, you may also contact Wouter Duetz directly by e-mail. This e-mail address can be found on the "about us" page.

Optionally you may provide us with additional details about your project; culture type & duration, throughput and desired culture volumes.

We are happy to assist you. We generally reply within a single business day.