▶Principles of shake-flask culture
The most common method of growing planktonic bacteria (i.e. those suspended in solution rather than attached to surfaces) in the laboratory is the shake or Erlenmeyer flask. This type of culture is referred to as a batch culture as all the components are used only for a single cycle of growth, in contrast to continuous culture and fed-batch cultures.
The inoculation process for batch cultures is not just a question of adding a few cells to the medium. Frequently the bacterial strain must be revived from storage, either from a lyophilized (freeze-dried) or frozen state. Bacterial and Archaeal cells will withstand freezing at -70°C for many years, provided that the solution they are stored in contains a compound that prevents the formation of ice crystals. Crystals of ice are thought to puncture the cell membrane, rendering the frozen culture useless. However, a solution of 50% v/v glycerol or 20–50% DMSO (dimethyl sulfoxide) can prevent this from happening. To bring the cells back from their frozen state, a rich undefined medium is used. This often takes the form of an agar streak, as this can simultaneously be used to check for the purity of the stored cells. An individual colony is then picked from the revival medium and inoculated into between 1 and 10 ml of the liquid medium to be used in the experiment. This first growth in a small-scale batch culture helps the strain to adapt to the medium conditions as well as to generate sufficient biomass for the next inoculation.
The 10 ml inoculum is then used to inoculate a larger, Erlenmeyer flask. Many microorganisms seem not to grow well unless the inoculum size is between 1 and 10% of the final experimental volume. The reasons for this are obscure and vary from culture to culture but may be related to one or more of the following factors:
- Carry over of an essential nutrient from the inoculum medium to the experimental flask.
- Some form of quorum sensing. Many Bacteria have mechanisms to detect the numbers of the same species in their immediate vicinity.
- Reduction in stress. On the macro level there would appear to be little difference between adding 1 ml inoculum to 100 ml of medium and adding 10 ml of inoculum to the same volume. On the microscopic scale, the inoculum and fresh medium are not perfectly and immediately mixed, so a larger inoculum may briefly form a gradient between established and new conditions, giving the individual cells slightly longer to adapt.
Once inoculated, the Erlenmeyer flask can be placed on a rotary shaker, held at constant temperature, and growth of the organisms can begin. How the organisms grow is subject to a number of growth-limiting factors. In batch culture with Erlenmeyer flasks, the greatest limiting factor is always the concentration of oxygen, which will frequently dictate whether the organism grows at all.
▶Entrainment of oxygen
Shake flasks have a limited culture volume based on the transfer of oxygen to the medium they contain. The concept of how much of a substance it is possible to move from one phase to another (in this case gaseous to aqueous) in a system is known as mass transfer. By trial and error it has been established that an aerobic organism will only grow optimally if the Erlenmeyer flask has a volume 10 times that of the medium it contains. Thus a 250 ml Erlenmeyer flask should ideally contain only 25 ml of medium. When growing Escherichia coli, this volume may be raised to as much as 50 ml, but this is only possible as E. coli is facultatively anaerobic. Practically, the maximum size of standard Erlenmeyer flask that can be used is 5 liters, so if culture volumes of more than 500 ml are required, alternative methods such as a simple stirred tank reactor must be used.
The oxygen mass transfer rate of shake flasks is limited because the system relies on the agitation of the surface of the medium to allow air to form small bubbles in the medium, and the oxygen to diffuse from those bubbles to the bulk medium. This entrainment of air can only happen at the surface of the medium, so if the level of the medium is too far up the conical flask, the surface area available is too small for mass transfer of oxygen to the medium beneath it. Entrainment of air can be increased by making the mixing of the liquid more vigorous, frequently by increasing the speed of agitation of the flask. Those organisms with a very high demand for oxygen can demand adapted flasks in which baffles in the sides of the flasks disrupt the smooth mixing of the medium further still.
▶Limitations of shake-flask culture
As outlined above, the main limitation of batch culture is the difficult balance that must be struck between the space available to incubate Erlenmeyer flasks and the oxygen mass transfer rate. In addition, batch culture should not be regarded as solutions of defined composition. Before the Bacteria are introduced into the medium, it may be possible to define all the components of the medium even in exact terms of the elemental concentration of carbon, nitrogen, phosphorus, sulfur, and so on. As soon as the organism starts to grow, the composition of the medium changes, and will continue to change until the organism stops growing. We assume that biomass increases and the carbon and energy sources decrease in a regular manner. However, microorganisms secrete a variety of small molecules into the medium as they grow, from protons to heterocyclic carbon compounds. Thus during batch culture, the pH as well as the concentrations of oxygen and many other compounds not only change but can fluctuate in an unpredictable manner.
To reduce the effect of pH fluctuations, the growth medium is buffered (normally with a phosphate buffer), but even so a change of one or more units of pH during batch culture is not uncommon. The onset of stationary phase is taken to be due to the lack of a suitable carbon source, but is frequently the result of the accumulation of toxic metabolites in batch culture. Despite these limitations, batch culture is a simple, quick, and for the most part reproducible method of growing small quantities of microorganisms in liquid culture.
For small-scale laboratory experiments, batch culture is ideal. However, relative to the time that the organism is growing, the time taken to prepare the equipment for another experiment (the down time) is long. The flask must be sterilized, cleaned, refilled with medium, autoclaved, and another inoculum prepared before another experiment can begin. This time can be minimized if the majority of the biomass and spent medium is poured away or removed by pumping, and fresh medium is added directly to the flask. The residual biomass serves as the inoculum for the next growth cycle. Although this is attractive in terms of reducing down time, it increases the number of interventions in the experiment, and thus the possibility of contamination increases as well. In situations where the culture itself is undefined, such as during enrichment of microorganisms with a particular property, then contamination can be seen to be a smaller problem. However, the possibility still remains that toxic metabolites will not be sufficiently diluted by the fresh medium to allow exactly the same growth parameters after feeding compared with the primary culture.