Shaking in microplate readers like James Bond
In fact, introducing a stirrer can contaminate the solution and lead to unwanted results. In general, shaking sample solutions might be used for various reasons including:
- Homogenisation of samples
- Increasing oxygen uptake in shaken cell cultures
- Increasing the rate of (bio)chemical reactions
- Introducing shear forces to break particles
Mixing solvents (e.g. after injection) might be the most common application for shaking. Nevertheless, there are many other assays where shaking is an essential part of the protocol. For example, maintaining aerobic conditions in a cell culture by permanent shaking to increase the oxygen uptake. Or in prion-related assays like the RT-QuIC assay which applies intensive shaking with up to 1100 rpm (rpm: rotations per minute) favouring aggregation conditions to shorten the aggregation lag-time.
Many shaking conditions for microplate-based assays were empirically adopted and not questioned. Nevertheless, there are some factors that should be kept in mind when shaking in microplates.
BMG LABTECH microplate readers have different shaking modes available including orbital, double-orbital and linear, which can be applied at different frequencies. Choosing the right combination of shaking mode, shaking frequency and microplate is essential to obtain reliable results. For instance, square well formats lead to a better mixing efficiency than round well formats since they act like baffles.
The well dimension and the filling volume also play an important role for mixing. Shaking a 1536-well microplate with half-filled wells at 100 rpm will not have any significant effect to the sample, whereas shaking a 6-well microplate with half-filled wells at the same speed will have a significant effect. Unfavourable conditions might occur if the 6-well plate is shaken at a higher frequency since the liquid inside the well might not be able to follow the external movement and lead to out-of-phase conditions.
The behaviour of the liquid towards the external movement is determined by its surface tension. The higher it is, the higher the shaking frequency must be to ensure thorough mixing. Aqueous solutions typically have a rather high surface tension and thus require higher shaking frequencies in order to move the liquid. However, there are other factors influencing the solutions’ behaviour towards external movement which are nicely discussed in a paper published in 2003 by Hermann et al.
Finally, the shaking settings that are used depend on the application, as mentioned at the beginning of this article. For example, breaking particles by shear forces will require higher shaking frequencies, while homogenisation will typically be done at lower shaking frequencies. The time that is needed to perform a given shaking action will also differ between the different applications. Cell growth assays (under aerobic conditions) are typically performed with permanent shaking while shaking for the purpose of homogenisation will be limited to a short period of time.
Taken together, James Bond seems to be aware of the fact that shaking is a non-invasive way of mixing cocktails without the risk of contamination by a stirrer. If your assay requires shaking, make sure to choose the correct microplate, the correct shaking mode and the correct shaking frequency. These can be easily set in BMG LABTECH’s Reader Control software when editing a test protocol.
Source: Robert Hermann, M. L., J. B. (2002, June 11). Characterization of Gas–Liquid Mass Transfer Phenomena in Microtiter Plates. Biotechnol Bioengineering, p. 9.