How Shaking Conditions Affect Microbial Cultures

Worldwide, many laboratories perform microbial growth assays in microplates to monitor multiple samples over time, avoiding tedious pipetting from a tube to a cuvette, reducing contamination risk, and enhancing assay throughput.

Turbidimetry measurements are often multiplexed with fluorescence or luminescence readings to follow marker expression.

The primary variables that impact microbial growth in culture are temperature, oxygen access (aerobic or anaerobic conditions), and medium composition. For aerobic strains, cultures are shaken to provide medium aeration, facilitating improved nutrient access and resuspending cells that may otherwise sediment. Moreover, maintaining the cells in suspension is critical to preserve the consistency of absorbance measurements.

Flask-grown cultures are generally shaken between 80 and 250 RPM during the assay. Since the smaller surface area in a microtiter plate ensures higher surface tension, microplate readers tend to shake samples at higher RPM, from 300 RPM to 1500 RPM.¹ This helps break the surface tension and allows sufficient medium agitation.

However, excessively vigorous shaking can cause cell damage, impacting growth or production.^2,3 In such cases, intermittent shaking may be an alternative to long-term continuous shaking in a microplate reader.

Brief (several seconds) but frequent (every few minutes) shaking could ensure sufficient medium oxygenation and aid cell resuspension, maintaining sample health.

Molecular Devices has partnered with researchers from the InBio.be group, who specialize in the development of various microorganisms for bioproduction. The aim of this partnership is to evaluate the impact of intermittent or continuous shaking on the growth of several prokaryotic and eukaryotic strains at differing temperatures or in different growth media.

This research demonstrates that specific microorganisms can grow well with intermittent shaking. Accordingly, the testing of different shaking intervals is suggested, which can be readily configured in a SoftMax Pro Workflow to develop an optimized workflow combining multi-mode measurements.

Advantages

• Easy configuration of personalized workflows with shaking for microbial growth experiments
• High-throughput microbial growth assays with ready-to-run protocols for greater confidence
• SoftMax Pro software streamlines growth curve data evaluation with automated Vmax computation

Materials

• SpectraMax^® iD3 Multi-Mode Microplate Reader (Molecular Devices, cat. #iD3) with
  • SoftMax^® Pro Software
• 96-well clear flat-bottom plates, sterile (Greiner cat. #655161)
• 96-well black flat-bottom plates (Greiner cat. #655077) used for fluorescence in Figure 5
• Bacterial or yeast strains grown by InBio.be (Ghent University) in their appropriate media. Strain names remain confidential and are referred to as prokaryote A, prokaryote B, and so on.

Methods

Various prokaryotic and eukaryotic strains were grown in their appropriate media following the SoftMax Pro workflows displayed in Figure 1. Absorbance values were normalized through the SoftMax Pro software data reduction menu using the following equation: Ln (OD₆₀₀/ODT0) by applying the formula Ln(!Lm1/Min(!Lm1)) as custom settings.

Automatic Vmax calculation was performed using the maximum number of data points to provide the highest squared correlation coefficient (R²). For in-depth information on kinetic data evaluation in SoftMax Pro, see the following application note: "Advanced kinetic analysis of a bacterial growth assay."

Figure 1. SoftMax Pro workflow protocols for continuous shaking (A) or intermittent shaking (B) with 10 seconds of agitation per minute and 50 seconds of idle time. Both types of kinetics were followed for 24 hours, taking a reading every 15 minutes. When GFP expression was followed, both in intermittent and continuous shaking, a fluorescence measurement was added after the absorbance reading (C). Shaking was done at 517 RPM with a 1.7 mm shaking diameter. Image Credit: Molecular Devices UK Ltd

Results

InBio.be assessed four prokaryotic and three eukaryotic species. All strains grew under continuous and intermittent shaking conditions. The shaking settings had different effects on the microorganisms depending on the species, temperature, and growth medium. Here, representative examples are shown; additional data is available upon request.

Prokaryotes A and B are aerobes and were grown at 28 °C. Prokaryote A exhibited comparable Vmax values and growth curves under continuous and intermittent shaking (Figure 2). Prokaryote B achieved a 30% higher Vmax value under continuous shaking; however, intermittent shaking significantly accelerated growth onset (eight hours with continuous shaking versus four hours with intermittent shaking) (Figure 3).

It should be noted that inoculation percentages were higher for the intermittent shaking plate. Under intermittent shaking, Prokaryote B exhibited a reduced lag phase and a smoother exponential phase. Both curves achieved the same final OD.

Figure 4 illustrates a sample eukaryote data set. While continuous shaking yielded a slightly higher Vmax (1.2-fold), the curves were largely overlapping and followed identical growth phases. Eukaryote A is a GFP-expressing strain.

Notably, GFP expression onset occurred earlier and exhibited a 1.2-fold rise in Vmax with intermittent shaking conditions (Figure 5) (note higher inoculation percentage as above). The increased GFP production early on could be due to the strain adapting more quickly to intermittent shaking conditions.

Another noteworthy case was eukaryote strain C, which exhibited growth in clumps. Under continuous shaking, this growth type exhibited numerous artifacts and significantly variable ‘zigzagging’ OD traces (Figure 5). Such irregular OD values are most probably artifacts.

Continuous shaking may encourage clumping, which could have impacted the optical path and/or contributed to air bubble formation if the solution contained a high amphiphilic-particle content (similar to a soap solution).

Under these conditions, calculating Vmax was impossible: the squared correlation coefficient (R²) for Vmax fit was between 0.56 and 0.92 for all replicates and the four different media evaluated under continuous shaking.

Intermittent shaking substantially enhanced signal stability, enabling the reliable determination of the sample growth rate (for the four media assessed, the average R² values for Vmax fit were 1.0, 0.99, 0.97, and 1.0).

Figure 2. Growth curves of prokaryote A at 28 °C, with continuous (orange) or intermittent (blue) shaking. Shown are the means of triplicate wells, with error bars representing standard deviation. Continuous: Mean Vmax = 3.751 mU/min, calculated using eight points with mean R² = 0.996; CV (Vmax) = 1.3%. Intermittent: Mean Vmax = 3.737 mU/minute calculated using eight points with mean R² = 0.996; CV (Vmax) = 3.8%. Image Credit: Molecular Devices UK Ltd

Figure 3. Growth curves of prokaryote B at 28 °C with continuous (orange) or intermittent (blue) shaking. Shown are the means of triplicate wells, with error bars representing standard deviation. Continuous: Mean Vmax = 4.028 mU/min, calculated using eight points with mean R² = 0.998; CV (Vmax) = 1.6%. Intermittent: Mean Vmax = 3.135 mU/minute, calculated using eight points with mean R² = 0.993; CV (Vmax) = 3.1%. Image Credit: Molecular Devices UK Ltd

Figure 4. Growth curves of eukaryote A at 30 °C with continuous (orange) or intermittent (blue) shaking. Shown are the means of triplicate wells, with error bars representing standard deviation. Continuous: Mean Vmax = 2.338 mU/min, calculated using 10 points with mean R² = 1; CV (Vmax) = 0.6%. Intermittent: Mean Vmax = 1.926 mU/minute, calculated using 10 points with mean R² = 0.999; CV (Vmax) = 0.5%. Image Credit: Molecular Devices UK Ltd

Figure 5. GFP expression in eukaryote A at 30 °C with continuous (orange) or intermittent (blue) shaking. Shown are the means of triplicate wells, with error bars representing standard deviation. Continuous: Mean Vmax = 4.541 mU/min, calculated using 10 points with mean R² = 0.997; CV (Vmax) = 0.6%. Intermittent: Mean Vmax = 5.469 mU/minute, calculated using 10 points with mean R² = 0.997; CV (Vmax) = 2.3%. Image Credit: Molecular Devices UK Ltd

Figure 6. Growth curves of clump-forming eukaryote C grown at 25 °C with continuous (orange) or intermittent (blue) shaking. Four different media were tested. Shown is one example out of a set of triplicate wells for one medium; similar results were obtained with other replicates and other media.

Average data for continuous shaking was not considered due to high variability among triplicates (CV up to 154%); Vmax could not be reliably calculated due to artifacts. Vmax was calculated for intermittent shaking using 20 Vmax points: Mean Vmax = 0.612 mU/min, R = 1, CV = 3.6%. Image Credit: Molecular Devices UK Ltd

Conclusion

This study has shown that different shaking conditions are required for different microorganism strains to achieve optimal growth. Shaking intervals can also influence marker expression and alter the microorganism’s growth phases.

Aerobic and rapidly growing strains such as prokaryote A may require more rigorous shaking, while slower-growing or more fragile strains such as eukaryote A may grow faster with intermittent shaking. Clients are advised to evaluate their individual strains for the best shaking intervals.

With the user-friendly SoftMax Pro software workflow editor, various shaking intervals can be easily programmed using the SpectraMax iD series readers. The advanced shaking upgrade available on the SpectraMax iD3s and iD5e readers enables further adjustment of the RPM and shaking diameter. This optimizes strain growth conditions, with rigorous shaking for long-term continuous shaking experiments.

In addition, SoftMax Pro software enables simple absorbance measurements (OD₆₀₀ values) for microbial growth and fluorescent marker expression in a single workflow, with completely automated data evaluation for extra convenience.

References and Further Reading

1. Duetz, W.A. (2007). Microtiter plates as mini-bioreactors: miniaturization of fermentation methods. Trends in Microbiology, 15(10), pp.469–475. DOI:10.1016/j.tim.2007.09.004. https://www.cell.com/trends/microbiology/abstract/S0966-842X(07)00172-2?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0966842X07001722%3Fshowall%3Dtrue.
2. Chung, C.-F., et al. (2020). Shaking Rate during Production Affects the Activity of Escherichia coli Surface-Displayed Candida antarctica Lipase A. Catalysts, 10(4), p.382. DOI:10.3390/catal10040382. https://www.mdpi.com/2073-4344/10/4/382.
3. Sakil Munna, Md., et al. (2014). Influence of Aeration Speed on Bacterial Colony Forming Unit (CFU) Formation Capacity. American Journal of Microbiological Research, 2(1), pp.47–51. DOI:10.12691/ajmr-2-1-7. https://pubs.sciepub.com/ajmr/2/1/7/index.html.

About Molecular Devices UK Ltd

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