Bacterial growth measurements on a microplate reader

Optimal bacterial growth

In the mid-17th century, Antonie van Leeuwenhoek was among the first to observe microbes, including bacteria, using a microscope of his own design.^1,2 Modern microbiology began much later in the mid-19th century powered by the development of many of the techniques needed to culture and study bacteria. These methods and innovations accelerated scientific progress and led to breakthrough discoveries across the life sciences. 

The study of bacteria has revealed many insights. Today, the importance of being able to measure bacterial growth rapidly and accurately cannot be underestimated. Bacteria are fundamental to life and play pivotal roles in health, industry, agriculture and the environment. Practical applications arising from the study of bacteria include the development of new diagnostics and drugs, food production and food safety, the discovery of new biofuels, as well as ensuring the quality of the environment through processes such as bioremediation and wastewater treatment. The measurement of bacterial growth underpins developments in biotechnology, drug discovery and emerging disciplines focused on synthetic biology. Understanding how bacteria grow impacts all these areas and has implications for some of the biggest challenges facing the planet including climate change and overcoming antibiotic resistance.^4,5 

In this blog we take a practical look at bacterial growth: what it is, what factors affect it, and how it can be measured using microplates. From there we look at some of the applications that can be realized on microbiology microplate readers through an understanding of bacterial growth as well as the challenges faced to optimize some of these applications. 

What is bacterial growth?

At the heart of bacterial growth is the process by which one bacterial cell becomes two. Most bacteria can quickly grow under the right conditions by the process of binary fission to rapidly yield a population of cells.

How fast can they grow? It’s a bit like asking for the top speeds of different cars. The doubling time for Escherichia coli, the laboratory workhorse, is around 20-30 minutes. Pseudomonas aeruginosa has doubling times of one to three hours depending on the nutrient environment used for study. Others like Mycobacterium tuberculosis may take up to a day to achieve the same milestone. Each bacterium has its own preferred conditions and nutrient requirements.  In comparison, the doubling times of human cells are typically longer. Eukaryotic cells may take about 24 hours or more to complete the cell cycle under optimal conditions in the body or in culture.

Bacterial growth stages

The life of bacteria can be divided into different stages of growth followed by a period of decline (Fig. 1).⁶ Three main phases are typically defined for growth when a small number of bacteria are introduced into a new growth medium or environment. In the lag phase, bacteria adapt to their new surroundings through metabolic and other changes, but they do not undergo cell division. In the exponential phase, bacteria begin to divide by binary fission and the population size doubles at a consistent rate specific to the bacterial species and environmental conditions. As mentioned earlier, the doubling time varies but is short for most bacteria and occurs within 20-60 minutes. After a period of rapid growth, proliferation typically hits a wall as environmental constraints like depletion of nutrients kick in. The equilibrium point, where cell division matches the rate of cell death, eventually gives way to a death phase. In some bacteria, mechanisms like spore formation or the ability to form biofilms can change the dynamics of the growth and death phases.The division into different life phases is a useful starting point to investigate bacteria. These controlled phases are most clearly observed under controlled laboratory conditions, but this framework has also proven useful to make research breakthroughs. Bacteria are extremely diverse and show considerable versatility in metabolism and genetics. The concept of growth phases can be mapped to their metabolism and different phases of genetic regulation. This is a way to look at enzyme synthesis and energy production, for example, during lag and exponential phases of growth.

Factors influencing bacterial growth

Environmental conditions significantly impact the life cycle of bacteria. In addition, scientists will often need to control growth conditions to make precise measurements of the phenomena they are interested in studying. At a fundamental level, factors like nutrients, temperature, pH and oxygen may need to be assessed for their impact on bacterial growth or controlled to make precise biological measurements in bacterial cell assays. Moisture and osmolarity also play important roles, while deep sea bacteria such as Photobacterium profundum SS9 even proliferate under extreme environmental conditions such as high hydrostatic pressure. 

In many cases, researchers use bacterial cultures to test their theories. Often, the controlled study of bacterial cells under different growth conditions permits researchers to study biology in a model system often without the complexity of events that take place in multicellular organisms. Irrespective, it is important to be aware of the conditions that promote or restrict bacterial growth. 

The factors that impact bacterial growth may not only be physicochemical but may also include the influence of other bacteria. Quorum sensing, for example, is a type of cell-to-cell communication in bacteria that depends on secreted chemical signaling molecules, bacterial cell density, and changes in gene expression. Quorum sensing has been shown to affect bioluminescence, symbiosis, expression of virulence genes, as well as antimicrobial resistance and the formation of biofilms in different bacteria. The stage of bacterial growth and cell density are therefore crucial parameters for measurements of these processes. You can read more about this type of bacterial communication in the blog Quorum sensing: how bacteria stay in touch. In addition, other bacteria may also produce toxins that limit bacterial growth of the organism under investigation.

Methods of measuring bacterial growth

Whether you are interested in pure or applied applications, measuring bacterial growth due to cell division over time or at least the cell density in your bacterial stock is often an essential prerequisite for downstream analyses. Broadly speaking, two types of techniques exist to measure bacterial growth: direct and indirect. Direct methods involve counting cells under a microscope, for example, often with a counting chamber or estimating viable bacterial cells from the number of colonies observed on an agar plate. Indirect methods measure bacterial growth by detecting changes in parameters that are influenced by growth, such as optical density at 600 nm (OD₆₀₀), light scattering, fluorescence or luminescence. 

Measurement of bacterial growth based on optical density (OD₆₀₀) is frequently used in many laboratories. The method comprises taking readings of bacterial samples over time that correlate with the number of organisms in a sample. OD₆₀₀ measures the amount of light scattering by a bacterial suspension and not its absorbance. For OD₆₀₀ the loss of light transmission due to scattering is measured. As bacteria grow and divide, they scatter more light as the turbidity of the suspension increases and the light transmission accordingly decreases. You can read more about measuring microbial growth using OD₆₀₀ in this blog from BMG LABTECH.

The nephelometer is a dedicated instrument to measure light scattering and is an alternative method to measure bacterial growth. Nephelometry measures the intensity of light scattered by (in this case) bacterial cells in a culture at an angle (typically 90 degrees) from the incident light (Fig. 2). Nephelometry offers higher sensitivity than absorbance-based methods and is particularly suited to measuring bacterial growth in samples with lower numbers of organisms. The NEPHELOstar Plus from BMG LABTECH is a dedicated reader that allows for early parts of the bacterial growth curve to be measured at high sensitivity as described in the application note Monitoring of microbial growth curves by laser nephelometry. In addition, fluorescence and luminescence measurements also offer ways to measure bacterial growth. Both methods are highly sensitive and can detect low numbers of bacterial cells. They include the use of fluorophores like green fluorescent protein (GFP) that can be expressed in the bacterial organism of interest. In the application note Expression of a stable green fluorescent protein mutant in group B Streptococcus: Growth, detection and monitoring with the CLARIOstar the use of a GFP biomarker was used to track the growth of bacteria in liquid media. The CLARIOstar^® was used to measure fluorescence, absorbance and fluorescence polarization simultaneously (Fig. 3). In this study, the growth medium contains compounds that autofluoresce at 515nm, which is the detection wavelength of GFP. Fluorescence polarization measures the slower rotation rates of high-molecular-weight species like GFP where a high proportion of incident plane polarized light is transmitted back to the detector. Lower-molecular-weight compounds like components of the growth media rotate more quickly and re-emit unpolarized light. Increases in fluorescence polarization therefore correlate well with the increased amounts of GFP that are expressed as cell numbers rise during bacterial growth. These measurements provide a highly sensitive alternative to conventional OD600 and fluorescence measurements which in this case were measured at the same time. Indirect measurement methods can also be used to detect or quantify bacteria. In the application note “InToxSa assay, for quantifying Intracellular Cytotoxic S. aureus Phenotypes”, propidium iodide was used to study the cytotoxic effect of intracellular S. aureus on a cell line. Other surrogate measurements for bacterial growth include metabolic activity assays (in particular, fluorescent resazurin assays and ATP measurements), genetic methods (quantitative PCR) and respiration measurements (assessing oxygen consumption or carbon dioxide production for example). Each offers greater speed and convenience than direct counting methods.

Some challenges in studying bacterial growth

Like most measurements, bacterial growth is prone to variability due to different factors. The accuracy, precision and reliability of results may be affected by biological variability (for example, cross-contamination with other bacteria or genetic mutations within bacterial populations), changes to environmental factors (e.g. temperature, pH, oxygen or other atmospheric changes, availability of nutrients), or variation in handling techniques (e.g. pipetting errors, insufficient mixing). Bacteria can quickly build up resistance to external agents or drug treatments due to their quick generation times which may also affect results. Evaporation can also pose challenges when studying bacterial growth. In this case, the use of sealers is recommended to minimize the evaporation of liquid from the wells of a microplate. Sealers can also help to prevent contamination of bacterial samples in a well or protect from potential unwanted effects of light and air.

In many cases, good experimental practice can help to minimize variability. This can include standardizing experimental protocols, providing consistent environmental conditions, maintaining and regularly calibrating instrumentation, or using appropriate controls and replicates in experiments. 

Applications and the use of microplates to study bacterial growth

As mentioned earlier in this blog, measurements of bacterial growth are at the centerpoint of many crucial applications in the life sciences. Bacteria are one of the most numerous life forms on the planet and inhabit almost all environments. Bacterial growth measurement is therefore crucial in many industries including the medical and healthcare, food, and environmental sectors. In the food industry for example scientists are looking at ways to develop food packaging with antibacterial properties. Likewise, researchers are interested in developing antibacterial coatings or protective biofilms for medical devices or implants used in biomedical interventions.

In environmental monitoring, bacterial growth measurements are being used to assess water quality, detect contamination and pollutants, as well as monitor bioremediation processes. In addition to the control of bacterial growth in areas like infection control (e.g. with antibiotics), food preservation and safety, and environmental waste management, bacteria are the workhorses of the biotechnology industry where they can be used to produce biofuels, enzymes and other biological products. These areas of biomanufacturing also depend on measurements of bacterial growth. Genetic engineering and the manufacture of proteins – for example in the production of new drugs – often involve bacteria as part of the production process. 

In the years ahead, synthetic biology is expected to deliver new ways to tackle some of the most pressing challenges in health, agriculture and biomanufacturing. In all cases the measurement of bacterial growth is a key part of the process.

As mentioned earlier, scientists have cultured bacteria for many years in liquid media, often using Erlenmeyer flasks, and have measured the proliferation of cells in a cuvette using absorbance measurements at 600 nm. The availability of microplate readers with temperature control and shaking options allows for kinetic studies of bacterial growth that require little or no intervention from the user over different durations of time. They also offer the capacity to make parallel measurements under a wide range of conditions using different detection modes. Multi-mode microplate readers therefore offer benefits in speed, reproducibility and scale for routine measurements of bacterial growth.

Some examples of bacterial growth measurements using microplates are shown in the BMG LABTECH application notes included in Table 1. These include examples that require an Atmospheric Control Unit (ACU).

BMG LABTECH Application Notes for bacterial growth

Atmospheric control

Bacteria that have no specific atmospheric requirements for growth will be happy in the standard environment of the microplate reader. Those requiring more specific oxygen or carbon dioxide conditions may show growth but not in a way that is typical or representative of healthy growth. This type of restriction could compromise experiments. The Atmospheric Control Unit from BMG LABTECH provides researchers with a system that uniquely enables control of both the oxygen and carbon dioxide concentrations in an independent manner. In the examples in Table 1, bacteria that grow well in BMG LABTECH microplate readers without the need for gas control include enteric Salmonella, Vibrio fischeri, and Corynebacterium glutamicum. 

More fastidious microbes require elevated CO₂ levels. A good example is the bacterium Neisseria meningitides which thrives in a 5% CO₂ environment. In the application note Growth of Neisseria meningitidis in a BMG LABTECH microplate reader with Atmospheric Control Unit (ACU) it was demonstrated that this fastidious bacterium could be grown well on a BMG LABTECH microplate reader coupled with an ACU to deliver 5% CO2 (Fig. 4). This set up proved highly effective to grow this bacterium when compared to other methods including a conventional CO₂ incubator.A wide range of applications can be supported by bacterial growth measurements on BMG LABTECH microplate readers which are also facilitated by the availability of the ACU (Table 1). These range from kinetic growth studies to investigations looking at quorum sensing or different approaches to interventions against antimicrobial resistance and biofilms. The control of bacterial growth is crucial in many areas related to health, industry, agriculture and the environment. In particular, the search for alternatives to classic antibiotics has become increasingly important as pathogenic strains are becoming increasingly resistant to commonly available antibiotics and new ways to control such infections are needed. In the application note Testing novel bacteriophages for antibacterial properties with a crystal violet biofilm quantification assay the use of bacteriophage was demonstrated as a way to combat pathogenic strains of Escherichia coli. In the study, a novel bacteriophage was shown to have promising antibacterial activity against several strains of antibiotic-resistant E. coli. A crystal violet biofilm assay was used to demonstrate the effectiveness of this type of approach to the control of unwanted bacterial growth (Fig. 5).

Shaking options

Infections with Salmonella bacteria are one of the leading causes of gastrointestinal disease. Accordingly, researchers are continually looking for new drugs to treat potential infections quickly or to prevent infections directly. In the application note High-throughput determination of bacterial growth kinetics using a BMG LABTECH microplate reader growth rates of biocide-resistant mutants of Salmonella and their parent strains were tested for their tolerance to different biocides. Growth curves were monitored in absorbance mode for 24 hours (Fig. 4). The study showed that exposure to different biocides selected for bacterial strains with increased tolerance to biocides and that there was no obvious impact on overall fitness.

Minimal inhibitory concentration

A crucial analysis parameter in the control of bacterial growth using bactericidal substances is the minimal inhibitory concentration (MIC). The MIC is the lowest concentration of an antimicrobial compound or molecule that inhibits microbial growth. Microplate readers can be used to determine the MIC by measuring the light signal (absorbance, fluorescence or luminescence) of samples in a microplate containing different concentrations of an antibacterial compound. The ability of a microplate reader to measure thousands of samples in a matter of minutes allows for quick and precise determination of the lowest concentration of inhibitor needed. In the application note High-throughput determination of bacterial growth kinetics using a BMG LABTECH microplate reader discussed earlier in this blog, MIC values were crucial parameters measured for the different biocides tested in the study.

Minimum bactericidal concentration

The minimum bactericidal concentration (MBC) is another quantitative metric that can be used to assess the effect of antibacterial agents on microorganisms. Whereas the minimum inhibitory concentration (MIC) indicates the lowest concentration needed to inhibit bacterial growth, the MBC indicates the lowest concentration needed to kill bacteria.

Conclusion

The demand for measurements of bacterial growth will continue to grow in the years ahead as new developments, technologies and applications emerge from science across the globe.  Further advances in detection technologies and innovation in specific assays for microplate readers should drive progress in measurements of bacterial growth.

BMG LABTECH solutions

What is the preferred BMG LABTECH microplate reader for specific needs and applications related to bacterial growth? Absorbance detection for the measurement of OD₆₀₀ is available on BMG LABTECH’s complete portfolio of microplate readers with the ultrafast spectrometer. The exception is the NEPHELOstar^® Plus which is a dedicated laser-based nephelometer for light scattering and turbidity measurements. 

BMG LABTECH also offers a range of multi-mode detection devices for sensitive fluorescence and luminescence measurements.

Bacteria require specific temperatures and aeration for maximum growth rates. To ensure optimal growth conditions, all BMG LABTECH readers offer accurate temperature regulation up to 45°C (optionally up to 65°C). Three shaking modes with adjustable speed up to 700 rpm (optionally to 1100 rpm) provide optimum aeration settings for your strain. Additionally, the VANTAstar, CLARIOstar Plus, the Omega series and the SPECTROstar Nano can be equipped with an extraordinary robust transport system for shaking 24/7 where required.

The VANTAstar, the CLARIOstar Plus, the Omega series and NEPHELOstar Plus can be combined with the Atmospheric Control Unit making them the preferred choice for different kinds of live cell assays including bacterial growth assays

Both the VANTAstar and CLARIOstar Plus further allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance in a single luminescence or fluorescence run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators^TM and different filter options.

All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus,  and PHERAstar® FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection. The VANTAstar can be equipped with a modular injection unit. The SPECTROstar Nano comes with a dedicated cuvette-port which can also be used to study bacterial growth over time in a cuvette-based approach.

Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturised assays, short measurement times, and offer considerable savings on materials and other resources.

The NEPHELOstar Plus offers turbidimetric measurements for the determination of bacterial growth at very high sensitivity. It can be used for example to study the early stages of bacterial growth.

References

1. Gribbin, J. Science: A history 1543-2001 (2002) London: The Penguin Press.
2. Nurse, P. What is life? Understand biology in five steps (2020) Oxford: David Fickling Books.
3. The Eighth Day of Creation: Makers of the Revolution in Biology (1979) Touchstone Books.
4. Ingraham, J. L., Maaløe, O., and Neidhardt, F. C. (1983) Growth of the Bacterial Cell. Sunderland: Sinauer Assoc.
5. Dean, A. C. R., and Hinshelwood, C. (1966) Growth, Function and Regulation in Bacterial Cells. London: Oxford University Press.
6. Panikov, N. S. (1995) Microbial Growth Kinetics. London: Chapman & Hall.