When culturing bacteria in a shaking incubator, several factors must be considered that will impact bacterial growth, including shaking speed, temperature, growth medium, and growth vessels. These will vary depending on your application, time course, bacterial strain, expression vectors, features of your incubator, and other factors. Shaking incubators continuously introduce dissolved oxygen into the culture medium, promoting aerobic bacterial growth. Choosing the proper settings and culture conditions is essential for producing the greatest yield while minimizing cell death and recombination events.
Bacterial cultures typically shake at 150-250 RPMs. A faster speed (350-400 RPMs) can be used to obtain greater cell density, as the higher speed offers increased aeration of the culture. The optimal speed will differ based on the orbital diameter of the shaking incubator. Most incubators have an orbital diameter of 19, 25, or 50 mm. Incubators with a smaller orbital diameter generally offer a higher maximum speed than those with a larger diameter. The oxygen transfer rate (OTR) is impacted both by the speed of agitation and the orbital shaking diameter. Higher speed and larger orbital diameter increase OTR, but the speed has a greater impact than the orbital diameter. Of course, there is an upper limit to the appropriate speed for growth, as too vigorous of agitation can result in cell death. You can read more about how orbital diameter and shaker agitation affect bacterial growth and how to calculate the optimal speed for your instrumentation in our previous blog post.
The optimal temperature for bacterial culture depends on the strain and plasmid being replicated. Though bacteria have a wide range of optimal growth temperatures, most bacterial strains utilized for replicating plasmids will grow best at 30°C or 37°C.It’s a good idea to check the recommended growth temperature for your plasmids. Lower temperature growth can limit the frequency of recombination. Cultures generally grow more slowly at 30°C than at 37°C and may require additional time.
New Brunswick™ Innova® 44 Series Incubator Shakers by Eppendorf are stackable to save space, and have a vast range of temperature settings, from Ambient -20°C up to 80°C. They come in a 1” or 2” orbit with speeds of 25 up to 200, 300, or 400 RPMs, depending on the model. Multiple accessories are available to accommodate vessels of most sizes.
If you need to grow very large cultures, the Jeio Tech ISF-7000 Series Incubated Shakers may be best suited. They have a high shaking speed up to 500 RPMs and a temperature range of Ambient -20°C to 80°C (for some models). The orbital shaking diameter is 25.4mm for the ISF-7100/R or 50.8mm for the ISF-7200/R. Though it can accommodate all sizes of growth flasks, these incubators can hold up to five 6-liter flasks at once.
The most common growth medium used is the Lysogeny Broth (LB) medium, often called LB Broth. However, there are a variety of alternatives to LB broth that offer greater yields for certain bacterial strains. Some of these alternatives include Terrific Broth, Tryptone medium, and SOC Medium, as well as derivatives of the LB Broth, including Lennox and Miller’s LB, which have higher salt levels than the original Luria Broth.
Shaking incubators can be fitted with various vessel holders to accommodate many sizes and shapes, as well as the number of vessels. Different vessels will impact the oxygen surface interface. The culture medium within the vessel should not exceed 10-25% of the vessel volume. Smaller vessels will have reduced surface area, inhibiting oxygen transfer.
Shaker Agitation Rate and Orbit Affect Growth of Cultured Bacteria - Manual from Thermo Fischer Scientific
Shaker Orbit – Revolving in Space Around the Samples? - Guide from Eppendorf
Inoculating a Liquid Bacterial Culture - Guide from Addgene
Millipore Sigma - Introduction to Microbial Media