Measuring Bacterial Growth Under Changing Culture Conditions

Valuable insights into microbial growth can be gained by measuring wavelength-specific absorbance of cultures over time.

Bacterial growth curves measured in 96-well microplates using a microplate reader enable simultaneous monitoring of multiple experimental conditions, while onboard injectors conveniently facilitate experimental additions whose effects on growth can be quantified instantaneously.

This article demonstrates the application of the SpectraMax^® iD3s Multi-Mode Microplate Reader with Advanced Shake and dual injectors for continuous measurement of E. coli culture growth over a 15-hour period. Plate shaking was performed between absorbance reads to maintain aeration and nutrient availability.

At a selected time point, injectors were used to introduce glucose or chloramphenicol to a subset of wells via injectors, with the results on growth observed as kinetic traces shown in SoftMax^® Pro Software.

Advantages

• Advanced shake settings allow users to dial in optimal microbe growth conditions
• Onboard injectors enable reagent addition with continuous growth data generation
• Workflow editor enables configuration of complex workflows

Materials

• E. coli, strain JM109
• Growth media, LB Broth (1X) (Thermo Fisher Scientific cat. #10855001)
• Glucose, 1 M stock (Fluka BioChemika cat. #49158)
• Chloramphenicol, 10 mg/mL in ethanol (Thermo Fisher Scientific cat. #J67273.AB). This stock solution was further diluted to 500 ug/mL prior to use.
• Clear 96-well microplate (Corning cat. #3599)
• QPix^® FLEX^™ Microbial Colony Picker (Molecular Devices)
• SpectraMax^® iD3s Multi-Mode Microplate Reader (Molecular Devices) with:
  • SoftMax^® Pro Software
  • Advanced Shake (optional feature)
  • Injector system with SmartInject^® Technology

Note: The SpectraMax iD5e Multi-Mode Microplate Reader also offers Advanced Shake and SmartInject technology and can be used for similar bacterial growth applications

E. coli Culture and Growth-Curve Generation

Using the QPix FLEX Microbial Colony Picker, E. coli strain JM109 was streaked onto LB agar plates. A single colony was then inoculated into 5 mL of LB broth and grown in a 15 mL culture tube with shaking for 14 to 16 hours at 37 °C, until reaching an optical density at 600 nm (OD₆₀₀) of about 0.9.

The overnight culture was subsequently diluted with fresh LB broth to an OD₆₀₀ of approximately 0.1 for the experiments.

Next, 190 μL of the E. coli culture was moved to each well B2 to G11 of a clear 96-well microplate, while LB broth was introduced to the wells along the microplate’s outer edges to reduce inner well evaporation.

Glucose was added to wells in columns four and five to a final concentration of 30 mM. The microplate was subsequently placed in the SpectraMax iD3s reader, and a kinetic read was started using a protocol with workflow configured in SoftMax.

Pro software as detailed below (see also Figure 1).

Injections of glucose, a nutrient, or chloramphenicol, a broad-spectrum antibiotic that inhibits bacterial protein synthesis, into replicate wells were scheduled to occur when cultures reached the mid-log growth phase.

Workflow Steps

1. The system runs 14 cycles for a total of 3.3 hours, composed of the following steps:
   1. The system reads absorbance at 600 nm (OD₆₀₀).
   2. Shaking is performed for 870 seconds in orbital mode with a diameter of 2.5 mm and a speed of 500 rpm (settings selected to match those of a laboratory shaker).
   3. The process is repeated from step (a).
2. Following completion of the 14 cycles, the system injects 6 μL of glucose stock into the wells of columns six and seven (final concentration 30 mM).
3. The system injects 3 μL of chloramphenicol stock into the wells of columns eight and nine (final concentration 7.7 μg/mL).
4. The system injects 15 μL of chloramphenicol stock into the wells of columns 10 and 11 (final concentration 36.6 μg/mL).
5. The system runs 57 cycles for a total of 14 hours, consisting of the following steps:
   1. The system reads absorbance at 600 nm.
   2. Shaking is performed for 870 seconds in orbital mode with a diameter of 2.5 mm and a speed of 500 rpm.
   3. The process is repeated from step (a).

Results

SoftMax Pro software was used to generate raw data on bacterial growth from the initial plating through the lag and log phases, and on responses to the addition of glucose or chloramphenicol. This information could be viewed as continuous kinetic traces at any time during the workflow (Figure 2).

The full workflow was configured to operate for 17 hours and 16 minutes, plus approximately one minute for the three injections. However, since the workflow run can be canceled at any point without losing any generated data, it was halted during cycle 46 of step 5 above, at approximately 15 hours, when distinct differences among the various experimental treatments were already evident.

For each tested growth condition, formulas were applied to the raw data using the Data Reduction feature in SoftMax Pro to determine the maximum OD₆₀₀ achieved during the growth period and the time at which it was reached for each microplate well.

In control wells, E. coli growth displayed a lag phase, followed by a log phase, attaining a maximum OD₆₀₀ of 1.19 at 12.6 hours (Figure 3A). Cultures to which glucose was added immediately before beginning the kinetic read exhibited a dimorphic (two-phase) growth curve, reaching a maximum OD₆₀₀ of 1.27 at 14.3 hours (Figure 3B).

When glucose was injected into cultures during the mid-log growth phase, growth became more continuous (monomorphic), reaching an OD₆₀₀ of 1.33 at 14.7 hours (Figure 3C).

Chloramphenicol injection reduced growth relative to control cultures, reaching a maximum OD600 of 0.75 at 8.2 hours, after which it declined. A final concentration of 36.6 μg/mL led to a slight increase compared to 7.7 μg/mL (Figure 3D). Figure 4 displays this growth-curve data reduced to maximum OD₆₀₀ (A) and time to maximum OD₆₀₀ (B).

Figure 2. Continuous kinetic traces of E. coli growth. Single representative wells of different growth conditions are shown to illustrate the variation in growth observed. Image Credit: Molecular Devices UK Ltd

Figure 3. Kinetic traces of replicate wells. A, control; B, glucose added at time zero; C, glucose added by injector at mid-log phase; D, high or low concentration of chloramphenicol (CHL) added by injector at mid-log phase of growth. Image Credit: Molecular Devices UK Ltd

Figure 4. Raw kinetic growth curve data with calculations applied using the data reduction function in SoftMax Pro software. Results for different culture conditions were plotted: A, maximum OD₆₀₀ reached during the kinetic read; B, time required (hours) to reach maximum OD₆₀₀. Image Credit: Molecular Devices UK Ltd

Conclusions

Glucose is a preferred carbon source when introduced into LB broth at the start of bacterial culture (e.g., E. coli). LB broth itself contains amino acids and peptides from tryptone and yeast extract that bacteria can also metabolize.

Growth proceeds in phase 1, when bacteria consume glucose first due to catabolite repression, which suppresses the genes for metabolizing other carbon sources. This results in an initial exponential growth phase.

Following glucose depletion, a lag phase of metabolic adjustment occurs, during which bacteria pause to reprogram gene expression (e.g., activate operons for amino acid metabolism). This creates a visible plateau or dip in the growth curve. Finally, bacteria enter phase 2, resuming their growth by using the amino acids and peptides in LB broth.

The second growth phase is usually somewhat slower than glucose metabolism. Known as diauxic growth, this two-phase pattern is well documented in E. coli grown in media containing both glucose and alternative carbon sources.^1,2

In this study, introducing glucose during the mid-logarithmic phase yielded a growth profile markedly different from the diauxic-like pattern observed when glucose was present from the beginning. The OD₆₀₀ trace followed a continuous sigmoidal trajectory rather than a biphasic curve with a transient lag.

These results show that cells already metabolically engaged with amino acids and peptides in LB were capable of incorporating glucose into central metabolism without needing a substantial regulatory reset.

The lack of a lag phase indicates that catabolite repression was less pronounced under these conditions, as the transcriptional and enzymatic machinery for using alternative nutrients was already active.

Metabolite research supports this interpretation: Nanchen et al. (2006) demonstrated that E. coli intracellular fluxes exhibit a non-linear dependence on growth rate, with cells in active log phase maintaining versatile metabolic states.³

Enjalbert et al. (2015) showed that acetate fluxes become substantial after glucose depletion; however, acetate accumulation decreases when glucose is added mid-growth, smoothing the metabolic transition.⁴

More recent systems-level evaluations (Succurro et al., 2019; Karlsen et al., 2023) highlight that population heterogeneity and the timing of substrate availability have a strong impact on whether a lag emerges.^5,6

Collectively, these findings show why adding glucose in mid-log phase produces a smooth S curve: the culture seamlessly integrates the novel carbon source into an already mixed substrate metabolism, preventing the characteristic pause of diauxic growth.

Chloramphenicol functions as a broad-spectrum antibiotic that binds to the 50S ribosomal subunit of bacteria, where it blocks the peptidyl transferase activity responsible for peptide bond formation.

The antibiotic exerts a bacteriostatic effect by blocking this essential stage in protein synthesis, pausing bacterial growth without directly causing cell death. However, it can be bactericidal against specific pathogens at elevated concentrations.

As anticipated, introducing chloramphenicol to E. coli in mid-log phase resulted in growth inhibition.

This was demonstrated by a decrease in maximum OD₆₀₀, which was attained more than eight hours earlier than in control wells. This growth pattern aligns with the mainly bacteriostatic mode of action of chloramphenicol. A small but noteworthy decrease in maximum OD₆₀₀ was observed with 36.6 μg/mL chloramphenicol compared to 7.7 μg/mL.

Summary

The effects of nutrient or antibiotic addition on bacterial growth can be readily visualized using the raw kinetic data acquired by the SpectraMax iD3s reader and the flexible workflow configuration enabled by SoftMax Pro software.

Onboard injectors facilitate the execution of more complicated experiments with timed reagent addition during growth data generation. The Advanced Shake feature enables selection of optimal shaking conditions.

References and Further Reading

1. Monod, J. (1949). The Growth of Bacterial Cultures. Annual Review of Microbiology, 3(1), pp.371–394. DOI:10.1146/annurev.mi.03.100149.002103. https://www.annualreviews.org/content/journals/10.1146/annurev.mi.03.100149.002103.
2. Sezonov, G., Joseleau-Petit, D. and D’Ari, R. (2007). Escherichia coli Physiology in Luria-Bertani Broth. Journal of Bacteriology, 189(23), pp.8746–8749. DOI:10.1128/jb.01368-07. https://journals.asm.org/doi/10.1128/JB.01368-07.
3. Nanchen, A., Schicker, A. and Sauer, U. (2006). Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures of Escherichia coli. Applied and Environmental Microbiology, 72(2), pp.1164–1172. DOI:10.1128/aem.72.2.1164-1172.2006. https://journals.asm.org/doi/full/10.1128/aem.72.2.1164-1172.2006.
4. Enjalbert, B., et al. (2017). Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway. Scientific Reports, 7(1). DOI:10.1038/srep42135. https://www.nature.com/articles/srep42135.
5. Succurro, A., Segrè, D. and Ebenhöh, O. (2019). Emergent Subpopulation Behavior Uncovered with a Community Dynamic Metabolic Model of Escherichia coli Diauxic Growth. mSystems, 4(1). DOI:10.1128/msystems.00230-18. https://journals.asm.org/doi/full/10.1128/msystems.00230-18.
6. Karlsen, E., et al. (2023). A study of a diauxic growth experiment using an expanded dynamic flux balance framework. PLOS ONE, 18(1), p.e0280077. DOI:10.1371/journal.pone.0280077. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0280077.
7. E. coli, strain JM109
8. Growth media, LB Broth (1X) (Thermo Fisher Scientific cat. #10855001)
9. Glucose, 1 M stock (Fluka BioChemika cat. #49158)
10. Chloramphenicol, 10 mg/mL in ethanol (Thermo Fisher Scientific cat. #J67273.AB). This stock solution was further diluted to 500 ug/mL prior to use.
11. Clear 96-well microplate (Corning cat. #3599)
12. QPix^® FLEX™ Microbial Colony Picker (Molecular Devices)
13. SpectraMax^® iD3s Multi-Mode Microplate Reader (Molecular Devices) with:

• • SoftMax^® Pro Software
  • Advanced Shake (optional feature)
  • Injector system with SmartInject^® Technology

Note: The SpectraMax iD5e Multi-Mode Microplate Reader also offers Advanced Shake and SmartInject technology and can be used for similar bacterial growth applications.

Acknowledgments

Produced from materials originally authored by Cathy Olsen, PhD, Senior Application Scientist at Molecular Devices, and Sushmita Sudarshan, PhD, Application Scientist, Assay Development at Molecular Devices.

About Molecular Devices UK Ltd

Molecular Devices is one of the world’s leading providers of high-performance life science technology. We make advanced scientific discovery possible for academia, pharma, and biotech customers with platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. From cancer to COVID-19, we've contributed to scientific breakthroughs described in over 230,000 peer-reviewed publications.

Over 160,000 of our innovative solutions are incorporated into laboratories worldwide, enabling scientists to improve productivity and effectiveness – ultimately accelerating research and the development of new therapeutics. Molecular Devices is headquartered in Silicon Valley, Calif., with best-in-class teams around the globe. Over 1,000 associates are guided by our diverse leadership team and female president who prioritize a culture of collaboration, engagement, diversity, and inclusion.

To learn more about how Molecular Devices helps fast-track scientific discovery, visit www.moleculardevices.com.

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