Contents
Why Baffles? - The Oxygen Dilemma
In shake flasks, oxygen transfer is often limited compared to controlled bioreactors due to smaller liquid surface areas and slower mixing. In bioreactors, controlled gassing helps maintain consistent oxygen levels, a technique that is not practical in shake flasks. Oxygen limitation can reduce microbial growth, alter metabolism, and lower product yields. While signs such as slow growth, unusual metabolite accumulation, or foam formation may be visible, oxygen limitation often goes unnoticed yet still has detrimental effects. For instance, E. coli produces more acetate via its secondary metabolism under oxygen limitation which not only reduces the final product yield but also lowers the pH favouring non-ideal conditions. Several strategies can improve oxygen availability in shake flasks but they may be incompatible with the bioprocess or already exhausted. Baffles, when used correctly and under the right conditions, reliably enhance oxygen transfer to the medium, supporting more efficient culture growth while maintaining a simple, scalable setup.
Different shake flask designs provide varying levels of oxygen transfer rates (OTRs). Baffled flasks consistently enhance OTRs compared to classical unbaffled (widemouth) flasks. Among baffled designs, differences exist too, e.g. corner-baffled flasks vs. flasks with bottom baffles. These designs also influence the OTR of the culture.
Figure adapted from: Running, J.A. and Bansal, K., 2016. Oxygen transfer rates in shaken culture vessels from Fernbach flasks to microtiter plates. Biotechnology and Bioengineering 113: 1729–173.
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Out of Phase Shaking
Baffles Make Waves
In a normal shake flask, the liquid generally moves smoothly in phase with the shaker, minimizing splashing, foaming, and shear stress. In baffled flasks, however, the liquid can move out of phase with the shaker motion due to the baffles disrupting the flow. This irregular movement can increase the risk of spilling, generate foam, and expose cells to higher shear stress, all of which can affect culture performance and reproducibility.
Read more: Büchs, J., et al., 2000: Out-of-phase operating conditions, a hitherto unknown phenomenon in shaking bioreactors
Foaming
Bubbles mean trouble
Foaming in shake flasks is generally undesirable, and it occurs more readily in baffled flasks due to the increased turbulence they create. Foam can block oxygen transfer and reduce the efficiency of mixing. Once formed, it is difficult to eliminate and can isolate the culture from the headspace, compromising mixing and reducing the accuracy and reproducibility of results. Persistent foaming can negatively affect culture growth and experimental outcomes, though adding antifoam agents can help control excessive foam and improve overall performance.
Read more: Vonester, D., et al. 2025, Foam Formation in Shake Flasks and Its Consequences
Spilling
Shake, Don’t Spill
Adequate headspace in shake flasks is crucial to prevent spilling during vigorous shaking. Baffled flasks create more turbulence than smooth-walled flasks, which improves mixing but also increases the risk of liquid splashing out. Having enough headspace can prevent overflowing, protecting both the sample and the incubator. Furthermore, splashing of liquid to the headspace and onto the flask closing can cause further problems, e.g., if the flask is closed with a cotton plug or membrane - these can get wet and clogged which would hinder gas exchange and severely decrease oyxgen transfer. It's particularly important to keep this in mind for high-volume or foamy cultures, where baffled flasks can otherwise become messy. Proper headspace ensures safe, efficient shaking while still taking advantage of the oxygen-transfer benefits of baffles.Reproducibility
Consistency? Not Always
Working with shake flasks can be challenging because no two flasks behave exactly the same. Variations in baffle shape or placement, combined with out-of-phase shaking, make results less consistent, especially in manually manufactured baffled flasks. These factors make it harder to accurately calculate the mass transfer coefficient (kₗa) and to predict culture performance. As a result, reproducibility between experiments can be low, and scaling up processes from flasks to bioreactors becomes more difficult. Careful standardization and awareness of these limitations are essential when relying on shake flasks for experimental data.
Side or Wall Baffles
Corning Baffled Flasks
Thomson Ultra Yield Flask (UYF)
Commercial Grade (e.g., Duran)
Hand-made (e.g., glass-blower)
Baffle Alternatives
Other Measures to Improve Oxygen Availability in Shake Flasks:
- Reduce Fill Volume (or use larger flask while keeping the absolute fill volume)
- Increase Shaking Speed
- Increase Shaker Throw/ Diameter
- Check Seal Type (Use cotton plugs instead of aluminium foil)
Multiparameter
Sensors
As the central piece of our DOTS Platform, the MPS enables effortless online monitoring of multiple parameters in shake flasks. Do you need to monitor biomass? Fluorescence? Dissolved Oxygen? Maybe you want it all at once? Do you want to add environmental parameters, like temperature and shaking speed? All this and more is now possible.
Dissolved Oxygen Sensors
The easiest way to measure dissolved oxygen (DO) in shake flasks! By just adding the factory-calibrated and pre-sterilized sensor to the medium and mounting the flask on top of the Multiparameter Sensor, DO can be measured effortlessly, providing high-density and real-time data. When combined with the Liquid Injection System (LIS), DO-based feeding can be realized! Available in different form factors, including pills and nanoparticles.
Liquid Injection System
As an integral part of the DOTS Platform, the Liquid Injection System (LIS) serves as a valuable tool for automated feeding in shake flasks. It opens the door to bioreactor-like bioprocesses within shake flasks, making fed-batch, biomass- or DO-based feeding, or automated promotor induction all attainable through its advanced capabilities.