Introduction
Today’s escalating crisis of antibiotic resistance means that determination of the Minimum Inhibitory Concentration (MIC) is increasingly important to ensure effective antimicrobial therapy 1,2. MIC is defined as the lowest concentration of an antibiotic that inhibits microbial growth and is an essential parameter for dosing studies and research to contain the emergence of drug-resistant strains 2.
Microplate readers can be used for the determination of MIC using OD600 measurements in low sample volumes 3,4. The use of microplates for the determination of MIC significantly increases throughput as multiple serial dilutions and replicates can be analysed concurrently. This high-throughput capability minimizes the variability of data and drastically reduces the time and labour associated with manual reading, ensuring a more robust and reproducible dataset. Moreover, automated data acquisition eliminates the subjective biases of conventional visual measurements of MIC and enhances accuracy.
In this application note, we demonstrate how a microplate reader can be used for the determination of MIC from OD600 measurements.
Assay principle
The OD600 measurement is a fast, non-destructive method to quantify the growth of microbes 5. As bacteria grow, light scattering increases due to the increasing number of cells in the culture (Fig. 1). OD600 measures light scattering through loss of transmission rather than absorbance per se which is typically non-existent. For the determination of MIC, OD600 is used to distinguish between concentrations of an antibiotic that are sufficient to inhibit microbial growth and those that do not.
Materials & methods
- 96-well plate, flat bottom, transparent, suspension plate (#650185, Greiner BioOne)
- Ampicillin (#HP62.1, Carl Roth)
- Gentamicin (#455310010, Thermo Fisher)
- LB broth (#10855021, Thermo Fisher)
- Escherichia coli (#6897, DSMZ)
- VANTAstar® (BMG LABTECH)
Experimental Procedure
Cultures of Escherichia coli (E. coli) were thawed and inoculated for 18 h at 37 °C in 200 mL Luria-Bertani (LB) broth. 100 µL bacterial suspension was transferred into 50 mL fresh LB broth to prepare the E. coli stock. Gentamicin and ampicillin were dissolved separately in H2O, filtered through a mesh with 0.22 µm for sterilisation and diluted in LB broth to prepare the antibiotic stock solutions. Two-fold dilutions of both antibiotics were prepared and 100 µL of each dilution was transferred into a 96-well plate (in quadruplicate). Next, 100 µL E. coli stock solution was transferred to each well. The final concentrations of the antibiotics were 0.1–50 µg/mL (Fig. 2). As a positive control for growth (GC), 100 µL E. coli suspension was mixed with 100 µL LB broth. The blank wells (B), which represent the assay background, included 200 µL LB broth only.
The accompanying lid with condensation rings was placed on the plate and OD600 of the standards and samples were analysed for 12 h with the following settings in the VANTAstar microplate reader for the determination of MIC.
Instrument Settings
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Absorbance, plate mode kinetic
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||
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Optic settings |
Wavelength |
600 nm |
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General settings |
Number of flashes |
22 |
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Settling time |
0.1 s |
|
|
Well Scan |
Spiral averaging |
2 mm diameter |
|
Kinetic settings |
Number of cycles |
144 |
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Cycle time |
300 s |
|
|
Shaking |
Double orbital, 900rpm, shake between readings |
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Incubation |
37 °C |
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Results & Discussion
GC wells containing only E. coli without the addition of an antibiotic substance showed a substantial increase in OD600 values over time. The culture showed a typical lag phase for growth. Samples treated with antibiotics showed a reduced increase in OD600 over time, depending on the concentrations of antibiotics used (Fig. 3).
The OD600 values after 12 h of incubation were compared to differentiate between preparations with and without bacterial growth. Therefore, all samples with an OD600 value that is 3 standard deviations (SD) above the mean value of the blank wells were categorised as positive (Table 1) and used for the determination of MIC.
Table 1: Categorisation of E. coli samples with and without growth based on the OD600 values after 12 h
Conclusion
Measurement of OD600 on a microplate reader provides a fast, reliable and objective method for the determination of the MIC value of an antibiotic substance. Microplate readers equipped with a UV-vis measurement option, allow the determination of bacterial growth in many samples in parallel, thereby providing a real-time, walk-away solution for the determination of MIC. BMG LABTECH readers permit microbes to be grown at a range of temperatures up to 65°C. The use of the optional Atmospheric Control Unit (ACU) also makes it possible to evaluate microbes, including fastidious organisms, with individual requirements for the concentration of O2 and CO2 gases in their culture environment.
The MARS Data Analysis Software simplifies the subsequent identification of samples with and without growth applying predefined templates. The underlying calculation streamlines the determination of MIC and eliminates subjective categorisation.
References
- World Health Organization, Fact sheet, Antimicrobial resistance, https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance.
- Kowalska_krochmal, B., Dudek-Wicher, R. (2021) The Minimum Inhibitory Concentration of Antibiotics: Methods Interpretation and Clinical Relevance. Pathogens. https://www.mdpi.com/2076-0817/10/2/165
- Ashour MB, Gee SJ, Hammock BD. Use of a 96-well microplate reader for measuring routine enzyme activities. Anal Biochem. 1987 Nov 1;166(2):353-60. doi: 10.1016/0003-2697(87)90585-9.
- Tiwari S, Nizet O, Dillon N. Development of a high-throughput minimum inhibitory concentration (HT-MIC) testing workfl ow. Front Microbiol. 2023 May 25;14:1079033. doi: 10.3389/fmicb.2023.1079033.
- How to optimize OD600 measurements, BMG LABTECH HowTo Note No.10, https://www.bmglabtech.com/en/howto-notes/how-to-optimise-od600-measurements/