Potential Errors Of Traditional Shake Flask Culture: A Barrier To Effective Optimization

Traditional Shake Flask

Shake flasks are the most common reaction vessels used in bioprocess engineering for screening and process development. Because of its simplicity, it can be used for a wide range of diverse activities. Despite its high practical value, the importance of engineering factors for shake flask cultivation has been underestimated for a long time.

Shake flasks are commonly used for medium optimization since they are simple to operate. Shake flasks have restricted oxygen supply and no control over crucial process factors like pH, compared to the production process, which is frequently performed in stirred-tank bioreactors.

The objective to keep in mind is that the primary practices might change the outcome of your shake flasks cultures. Here are some of the mistakes which might stand as a barrier to getting the most out of your results. This guide will explain why these things are considered as potential errors against your shake flask culture.


A conventional Erlenmeyer flask’s filling volume is 20% of the overall volume, therefore a 250 mL shaking flask will hold 100 mL of culture. This is already higher than the fill volume, resulting in improved oxygen transport (10-15%). Filling above 25% will suppress aerobic culture growth while also increasing wear on shaker bearings due to the increased height of the load.

The purpose of shaking with an Erlenmeyer flask or T75 flasks is to ensure thorough mixing and to increase the surface area available for oxygen transfer. The shaking movement might partially distribute a lesser volume along the flasks’ sidewalls. A massive amount of the culture will not move from the flask’s center if the fill volume is too high, resulting in a deeper liquid level and a lower surface area for gas transfer.

This additional capacity also raises the height of the load on the shaker platform for large T175 flasks. The forces exerted vary proportionally to the square of the height, so doubling the height and the maximum weight or speed possible is reduced by one-fourth. It is better for both the culture growth and the shaking mechanism to avoid overfilling. (Allman)


There must be a temperature difference between the room temperature and the minimum control temperature for any temperature control system to perform efficiently. If cooling is unavailable, a temperature controller’s only option is to turn off the heating if the actual value exceeds the set point.

The impact on shake flask culture can be significant. For mammalian cells, even a single degree of overheating can be enough to kill them or severely limit their survival and growth.

For insect cell cultures, this is already too high, and for other yeasts, it’s on the verge of being too high. In many parts of the world, summer temperatures of 30 °C or higher are nearly certain. Unless the laboratory has a very efficient air conditioning system, cooling is basically a requirement for all cultures except thermophilic cultures. It will be more efficient and less expensive to cool only the incubation chamber.


Taping a reference thermometer to an incubator shaker’s window isn’t a “sin” in itself, but believing the value is. The thing to keep in mind is that the reading does not represent the temperature of the incubation chamber. It’s a combination of the air temperature inside the chamber reaching the door and the ambient temperature seeping through the clear panel.

If you believe this reading is accurate, you may decide to adjust the control setting by a degree or more to get the thermometer reading closer to the desired growth temperature, which may harm the culture.

An optional external temperature sensor put in a flask on the shaking platform can be employed for accuracy. To achieve optimal stability of the reading, this must be filled to a similar volume as the growing flasks and placed in a desirable position.


These flasks can easily be available in the market. If you are looking for flasks, we at Molecular Biology Products have a wide variety of them along with a T25 flask.

Work Cited

Allman, Tony. “The 7 “Deadly Sins” of Shake Flask Culture.” INFORS HT, 6 May 2021, Accessed 6 January 2022.

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