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Shake Flasks – More Than Just Filling Volumes

Ines Hartmann Lab Academy

Shake cultures are grown in special vessels, also known as Erlenmeyer flasks. In contrast to other lab vessels, shake flasks have a characteristic shape to allow a good shaking performance without spilling liquid. Learn more about differences in design and material and its influence on aeration.

Shaking bioreactors - also named Erlenmeyer or simply shake flasks - were introduced in the beginning of previous century. With a capacity from 25 mL to 5 liters they offer the flexibility for a wide range of experimental purposes from screening and expansion, up to media design and early process development. They are economic in price and suited to cultivate bacteria, yeast and fungi, as well as plant and animal cells in suspension. Although shake flasks do not need specific training to handle them, there are certain things to consider for efficient cultivation and there are different vessel types which suit different applications.

Flask material – glass versus plastic
Flasks are available in either glass or plastic. For classic microbiology applications reusable autoclavable glass Erlenmeyer are appropriate in most cases. Also, when oxygen transfer is critical, it can be beneficial to work with glass, as glass as hydrophilic material supports the formation of the liquid film formation that is responsible for the oxygen transfer. In comparison to that, plastic is, if not specifically treated, hydrophobic [1]. When contamination matters, e.g. when handling sensitive cultures or production steps, single use sterile disposable flasks with 0,2 µm filter vented caps offer utmost convenience and safety. Different plastic materials are on the market depending on the application and on personal preference. From highly resistant Polypropylene (PP) for microbiology applications up to optically clear materials with focus on mammalian cell culture applications, like Polycarbonate (PC) or polyethylene terephthalate glycol (PETG).

Flask design – Special shapes and baffles
The aim of shaking cultures is to increase aeration and availability to nutrients and to prevent sedimentation. The typical Erlenmeyer flasks has a conical body with a wider base and a cylindrical neck. A variety of different flask designs exist, with wide or narrow necks, with or without baffles. Special designs are available to further improve gas exchange, like the large volume Fernbach flask with a wider base and hence a large area for oxygen transfer or the disposable Ultra Yield™ and Optimum Growth Flasks™ with optimized shape to maximize the surface-to-volume ratio [2].
Baffled versus non-baffled: In non-baffled flasks a uniform regular swirling liquid flow is generated with a well-defined and predictable geometry [2]. In comparison to that, this swirl is disrupted on purpose in baffled flasks by implementing defined cavities in the bottom area. This ‘breaking the swirl’ design improves the aeration of the culture and can be beneficial to use when the culture’s oxygen requirements are high. Beside an improved aeration, baffled flasks can also be useful when handling viscous cultures, e.g. filamentous fungi to prevent spore aggregation or culture pelleting [3]. On the other hand, there is a higher risk of foam formation which may hinder the oxygen transfer. Also baffled flasks give considerably more variable results than un-baffled due to the abrupt disruption of the swirl and more chaotic flow behavior [2].

Erlenmeyer Flask designs (a) wide-neck (b) narrow-neck (c) non-baffled (d) baffled design (e) Fernbach flask
https://handling-solutions.eppendorf.com/cell-handling/bioprocess/processes-and-applications/detailview/news/its-not-just-about-size-talking-about-shake-flasks-and-bioreactors/​​​​​​​

Flask closures - From traditional cotton to vented caps.
To prevent contamination of the cultures, different closures for flasks are available. The closure should prevent contamination but must facilitate enough aeration of the cultures. Closures range from traditional plugs, over metal caps and silicone sponge up to single-use filter caps. The rate of oxygen transfer through the closure depends on the diffusion coefficient for oxygen in the material, the width of the neck opening, and the stopper depth [1]. If contamination is an issue, cotton plugs should be avoided, as spillages (e.g. when baffled flasks are in use) or condensation vapor may moisten the cotton and result in cross-contamination. Wetting the flask closure in general should be avoided, not only to reduce cross-contamination, but also to not reduce gas-permeability. This also applies also for non-hydrophobic filter materials, which may soak up the moisture too. The same type of closure should be selected for parallel experiments to no have differences in gas transfer rates.

What filling volumes are recommended?
The higher the culture need for aeration, the lower the fill volume to be selected. As a rule of thumb, a fill of 1/5 of nominal flask capacity should not be exceeded for microbial cultures, e.g. 100 mL for a 500 mL Erlenmeyer flask. If maximum oxygen transfer is required e.g. for long fermentations of high oxygen consuming strains, the filling volume should be reduced to even as little as 10 % with the rotational speed increased as much as the culture resistance to shear stress allows. For shear sensitive mammalian cultures higher fill volumes between 30 - 40 % are common with lower shaking speeds ≤ 150 rpm applied. Specific flask types targeting high yield protein or plasmid expression evolved over the last years, like the above mentioned Ultra Yield™ and Optimum Growth Flasks™, both with optimized surface-to-volume ratio and bottom baffles for better oxygen transfer allowing higher filling volume [2].

References:
[1] Pauline M. Doran, Bioprocess Engineering Principles (Second Edition), Chapter 10 - Mass Transfer, 2013
[2] htslabs.com
[3] J. Büchs / Biochemical Engineering Journal 7 (2001) 91–98
[4] Filamentous Fungal Cultures – Process Characteristics, Products, and Applications Hesham A. El-Enshasy, in Bioprocessing for Value-Added Products from Renewable Resources, 2007