In microbiology, the precise quantification of bacterial growth forms the basis for numerous areas of research – from basic research and pharmacology to food microbiology. While traditional measurement in cuvette spectrophotometers is time-consuming and prone to error, microplate readers have revolutionised analysis. They enable automated high-throughput analysis under controlled conditions.
Principles and methods of measurement
Bacterial growth analysis is based on detecting physical and chemical changes in the culture medium during cell division.
Optical density (OD600)
The most widely used method is light-scattering measurement, often incorrectly referred to as absorption. At a wavelength of 600 nm (OD600), most cellular components absorb very little light; instead, the bacterial cells scatter the incident photons. The higher the cell concentration, the less light reaches the detector.
- Indirect determination: The OD correlates linearly with the cell count as long as the suspension is not too dense (usually up to an OD of 1.0).Â
- Advantage: It is a non-destructive method in which the samples can remain in the reader throughout the entire growth cycle.
Analysis of cell growth curves
Through continuous measurements at intervals (e.g. every 10 to 15 minutes), the reader’s software generates precise growth curves. These allow the kinetic parameters to be determined mathematically:
- Lag phase: adaptation of the bacteria to the medium
- Exponential phase (log phase): phase of maximum division rateÂ
- Stationary phase: nutrient deficiency or accumulation of metabolites stops net growth
- Death phase: decline in viable cell count
Multi-mode detection: Beyond OD
Modern devices are often so-called multi-mode readers. These offer additional detection technologies for deeper physiological insights:
- Fluorescence: Use of reporter genes such as GFP (green fluorescent protein) to track the expression of specific genes during growth.Â
- Luminescence: Measurement of ATP concentration (as a marker for metabolic activity) or luciferase activity.
- TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer): For investigating complex protein-protein interactions in bacteria.
Environmental control: The key to reproducibility
A key advantage of specialised equipment is integrated environmental control. Bacterial growth is particularly sensitive to physical fluctuations. One point is temperature control: Constant incubation temperatures (e.g., 37°C for human pathogens or 30°C for environmental microbes) are essential to minimise variability between wells.
To prevent bacterial sedimentation and ensure sufficient oxygenation of the medium, readers offer various shaking modes (linear, orbital, or double orbital). This ensures a homogeneous suspension and uniform growth in all samples.
For laboratories that place the highest demands on these parameters, specialised microplate readers offer advanced options for long-term incubation and precise atmosphere control (e.g. O2/CO2 regulation).
Key areas of Application
Antibiotic screening (MIC determination)
Microplate readers are the gold standard for determining the minimum inhibitory concentration (MIC). By testing different antibiotic concentrations in parallel in a 96- or 384-well plate, it is possible to determine precisely the concentration at which no significant growth occurs.
Phage interactions
The dynamics between bacteriophages and their host bacteria can be observed extremely well. A sudden drop in OD in a growing culture indicates the lysis of bacteria by phages, which is of great importance for phage therapy research.
Biofilm analysis
Bacteria in biofilms behave differently than planktonic cells. Microplate readers support screening for biofilm-forming strains, often combined with staining methods (e.g. crystal violet staining), which are quantified after washing the plate in the reader.Â
Contamination monitoring
In the pharmaceutical industry, readers are used to detect endotoxins (LAL test). Kinetic measurement of colour development or turbidity enables highly sensitive purity testing.
Advantages over manual methods
Switching from individual measurements to microplate readers offers significant advantages:
- High throughput: Analysis of hundreds of samples simultaneously in a single run.Â
- Automation and time savings: Walk-away systems allow researchers to collect data overnight without having to manually remove samples.Â
- Reduced risk of contamination: Since the plate can remain sealed during measurement, the risk of foreign contamination is significantly reduced.Â
- Data integrity: The integrated software automatically calculates doubling times and growth rates, eliminating human transcription errors.
Practical Applications
To achieve optimal results, users should ensure they select the correct plate type (e.g. clear bottoms for OD measurements, black walls for fluorescence). It is also important to correct for the path length effect, as the OD in the microplate reader depends on the fill volume in the well – in contrast to the fixed layer thickness of a standard cuvette (1 cm).
The use of microplate readers is no longer a luxury, but a necessity for any microbiological laboratory that relies on accurate, scalable and reproducible data.
References
- Beal, J., Farny, N. G., Haddock-Angelli, T., Selvarajah, V., Baldwin, G. S., Buckley-Taylor, R., Gershater, M., Kiga, D., Marken, J., Sanchania, V., Sison, A., Workman, C. T., & iGEM Interlab Study Contributors (2020). Robust estimation of bacterial cell count from optical density. Communications biology, 3(1), 512. https://doi.org/10.1038/s42003-020-01127-5
- Ergin, E., Dogan, A., Parmaksiz, M., Elçin, A. E., & Elçin, Y. M. (2016). Time-Resolved Fluorescence Resonance Energy Transfer [TR-FRET] Assays for Biochemical Processes. Current pharmaceutical biotechnology, 17(14), 1222–1230. https://doi.org/10.2174/1389201017666160809164527
- Kowalska-Krochmal, B., & Dudek-Wicher, R. (2021). The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance. Pathogens (Basel, Switzerland), 10(2), 165. https://doi.org/10.3390/pathogens10020165
