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Description of Methodology for Testing the Synergistic and Additive Effects of Antibiotics in Vitro Cover

Description of Methodology for Testing the Synergistic and Additive Effects of Antibiotics in Vitro

Advancements of Microbiology
Volume 63 (2024): Issue 4 (December 2024)
By: Paweł Z. Kmiecikowski,  Aniela Gabriel,  Dagmara Depka and  Tomasz Bogiel  
Open Access
|Jan 2025

Figures & Tables

Fig. 1.

Broth microdilution method – preparation of a concentration pattern. A– add Mueller-Hinton broth. B– prepare a series of microdilutions of the first antibiotic. C– prepare a series of microdilutions of the second antibiotic. D– add antibiotics together. E– re-add of Mueller-Hinton broth.
Broth microdilution method – preparation of a concentration pattern. A– add Mueller-Hinton broth. B– prepare a series of microdilutions of the first antibiotic. C– prepare a series of microdilutions of the second antibiotic. D– add antibiotics together. E– re-add of Mueller-Hinton broth.

Fig. 2.

The “time-kill” method – a series of dilutions. A– prepare six 96-well plates (t0, t1, t2, t4, t6, t24) with 0.9% NaCl B– add the bacterial suspension to test tubes 1st–4th. C– transfer the suspension portion to the appropriate plate at predetermined times. D– obtain a dilution of 1:10. E– transfer a portion of the suspension to the plates using the “drop plate” method.
The “time-kill” method – a series of dilutions. A– prepare six 96-well plates (t0, t1, t2, t4, t6, t24) with 0.9% NaCl B– add the bacterial suspension to test tubes 1st–4th. C– transfer the suspension portion to the appropriate plate at predetermined times. D– obtain a dilution of 1:10. E– transfer a portion of the suspension to the plates using the “drop plate” method.

Fig. 3.

CombiANT method – the insert and experimental protocol. A– the insert design: antibiotic reservoirs (a-c), interaction imaging area (d). B– the assay protocol: „The insert is loaded by adding 0.5 mL liquid agar (60°C) containing antibiotics into the reservoirs. The prepared inserts can be stored at 4–8°C. To activate an insert, add a second layer of 25 mL agar to enclose the insert and fill the plate, thereby permitting diffusion of the antibiotics to the agar surface and the reservoir periphery. After solidification, a bacterial cell suspension of 0.5 McFarland is inoculated on the agar surface using a sterile cotton swab and exposed to the antibiotic gradient landscape. The finished plates are incubated at 37°C, and stable zones of growth inhibition are established within 16–24 hours.” (Fatsis-Kavalopoulos et al. 2020)
CombiANT method – the insert and experimental protocol. A– the insert design: antibiotic reservoirs (a-c), interaction imaging area (d). B– the assay protocol: „The insert is loaded by adding 0.5 mL liquid agar (60°C) containing antibiotics into the reservoirs. The prepared inserts can be stored at 4–8°C. To activate an insert, add a second layer of 25 mL agar to enclose the insert and fill the plate, thereby permitting diffusion of the antibiotics to the agar surface and the reservoir periphery. After solidification, a bacterial cell suspension of 0.5 McFarland is inoculated on the agar surface using a sterile cotton swab and exposed to the antibiotic gradient landscape. The finished plates are incubated at 37°C, and stable zones of growth inhibition are established within 16–24 hours.” (Fatsis-Kavalopoulos et al. 2020)

Fig. 4.

Assessment of the synergistic effect of antibiotics by the method of constant coefficients.
Assessment of the synergistic effect of antibiotics by the method of constant coefficients.

Fig. 5.

Assessment of the synergistic effect of antibiotics by cross-method.
Assessment of the synergistic effect of antibiotics by cross-method.

Fig. 6.

Evaluation of the synergistic effect of antibiotics by the MIC:MIC ratio evaluation.
Evaluation of the synergistic effect of antibiotics by the MIC:MIC ratio evaluation.
DOI: https://doi.org/10.2478/am-2024-0017 | Journal eISSN: 2545-3149 | Journal ISSN: 0079-4252
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Language: English, Polish
Page range: 199 - 222
Submitted on: Aug 1, 2024
Accepted on: Nov 1, 2024
Published on: Jan 29, 2025
Published by: Polish Society of Microbiologists
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
antibiotic resistance,
antibiotic synergism,
checkerboard test,
time-kill test
Related subjects:
Life sciences,
Microbiology and virology

© 2025 Paweł Z. Kmiecikowski, Aniela Gabriel, Dagmara Depka, Tomasz Bogiel, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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