Shake flasks are very popular as they are easy to use and cost effective. To achieve statistically reliable and robust results reproducibility is crucial. This refers to the reproducibility of shake flask cultivations within the same experiment as well as to sequential experiments. Only if results are reproducible, they can be trusted scientifically and used for screenings, process developments and basic research.
Almost every scientist is probably familiar with the game, and sometimes despair , of looking for the cause of different results of replicates. Although experiments were started under the same conditions, the results of the cultivation drifted apart. It gets even more complicated if the vessel size changes and processes are scaled up.
There will always be processes for which reproducibility is easier than for others. For example E. coli cultivations are highly reproducible. Cultivations with filamentous fungi or plant and animal cell cultures can be trickier. But some basic rules to increase reproducibility apply for all of them.
Let's look at a cultivation step-by-step to understand which aspects are important for good reproducibility.
- Medium Preparation
When considering media, it is important to choose one in which components are well defined. While readily available, complex media, also known as general-purpose or basic media, can have different compositions depending on the batch. This is a factor that can't be controlled by the scientist in the lab and could have a serious impact on the outcome of your experiment. Defined media, on the other hand, consist of ingredients whose chemical formulas are known. But if a defined minimal medium is used it should be produced carefully.
Ingredients should be added in the same sequence and be autoclaved in a controlled manner to ensure consistency. For example, if sugar solutions are autoclaved, caramelization can occur depending on temperature, pressure, and length of the autoclave processes. Or if phosphate is added, it can precipitate with other medium ingredients. Important factors to consider include: temperature, time of mixing, time of autoclaving, age of chemicals and solutions, chemical distributor and batch, clean mixing and storage vessels, quality of distilled water and precise scaling of ingredients. *Note that this list is not complete. - Inoculation and Preculture Procedure
To start a cultivation, the prepared medium is inoculated with cells. Cells can be picked from an agar plate, stock cultivation, or cyro cultures. The number of cells picked, in which stage the stock cultivation is, or how “old” those cultures are can differ extremely. Additionally, the organisms have to adjust to the conditions in the shake flask which can also take a toll on initial cell health and viability.
To overcome these issues, often one or two precultures are performed before doing the main culture. Through the precultures, the organisms are adapting to the medium and shaking conditions. Therefore, of course the same conditions should be applied for pre and main culture.
Monitoring biomass growth or oxygen levels can assist in identifying the ideal time to transfer precultures to a main culture. Often the late exponential phase is chosen for this purpose as the cells are then considered to be adapted, fit and have with a high cell density. By following these steps, the organisms in the main culture always start off at a similar point of fitness and adaptation to the medium and shaking conditions. These steps help to increase reproducibility of experiments performed not at different times. - Culture Procedure
Once the media has been prepared, the cells inoculated and a preculture performed, the main cultivation can be then started. Here a sample from the preculture is taken, the OD is determined offline and the necessary amount of medium is calculated to start of the cultivation with a specific OD, such as 0.5. Also, the medium needs to be prepared and distributed to the flasks. This takes time! Some strains are quite robust and won't be affected if the cultivation is standing around for a period of time. For other organisms, it’s crucial to transfer them as fast as possible to the main culture and begin shaking in in the incubator. With this in mind, a standard procedure helps to do those steps in a shorter time and improve reproducibility.
If several flasks are used in the main culture, a master mix should be prepared, inoculated and distributed to the shake flasks. As particles and cells can sediment, this master mix should be homogenously mixed before aliquoting it to the flasks. This ensures that each flask contains the same starter cultivation and medium composition.
During the incubation, cultures are shaken with a specific shaking frequency, shaking diameter, temperature, humidity and filling volume. Mind that e.g. the set temperature displayed by the shaker might not be precise. This should be controlled, if different shaker are used for experiments.
Also taking samples is a crucial factor for reproducibility. Each time a sample is taken, the shaker is stopped and opened which changes the temperature, the mixing, and oxygen supply. If samples are needed, they should be taken as fast as possible to reduce the interruption to the cultivation. Also each flask should only be sampled once. Taking a sample reduces the filling volume which changes the oxygen supply. And the oxygen supply can have a significant impact on growth and product synthesis. A flask from which a sample is taken can therefore no longer be compared directly to an unsampled flask.
If possible, taking samples should be avoided completely or reduced to a minimum. Therefore, online analytics are essential. With online determination of e.g. pH, biomass growth, fluorescence or dissolved oxygen critical events during the cultivation can be detected and indicate the times that samples should be taken. On the other hand, side online analytics also enable quality control and the success of reproducibility. Therefore, online measurements in shake flasks became more and more popular over the last 20 years.
By looking at the factors influencing reproducibility explained above it becomes clear that the scientist preparing and performing the experiments can be the game changer. The scientist should work precisely, be observative and concentrated, always aware of the pitfalls. But human error still does occur! The final important step to keep in mind is that documentation and discussion of those variations is crucial to evaluate later results and find the cause for seemingly non-reproducible data.
So remember, there are several ways to increase reproducibility in shake flasks. And the more online analysis is possible in shake flasks, the better the control and reproducibility is possible.
