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Antimicrobial Synergy Testing/Checkerboard Assay

The antibacterial synergy test/checkerboard test is an experimental method used to evaluate the interaction of two antibacterial test products. In this assay, the MIC and MBC values of the test compound are used alone, and combined with the MIC and MBC values of each bacterial strain evaluated to calculate the accumulation.

Synergy measurements by checkerboard analysis can be used to determine the change in antibacterial efficacy of a combination of antibiotics relative to their individual activity. The comparison is then calculated as a fractional inhibitory concentration (FIC) index value. FIC index value shows the combination of antibiotics with the greatest change compared to the MIC of a single antibiotic.

The figure below shows an example of a checkerboard test method in which the synergistic activity of kanamycin and ampicillin is determined.

High Throughput Screening (HTS) ServicesFigure 1. Checkerboard method showing the synergy effect on Pseudomonas aeruginosa.

Our Procedures

  • Before the test, a stock solution of each drug and two consecutive dilutions were prepared, leading to at least double the MIC.
  • A total of 50μl of Mueller-Hinton broth was dispensed into each well of the microdilution plate.
  • The first antibiotic in the combination is diluted sequentially along the ordinate, while the second drug is diluted along the abscissa.
  • An inoculum equal to 0.5 McFarland turbidity standard was prepared from each Pseudomonas aeruginosa isolate in Mueller-Hinton broth.
  • Inoculate 100μl of 5 x 105 CFU/ml bacterial inoculum in each microtiter well, and incubate the plate at 35°C for 48 hours under aerobic conditions.
  • The resulting checkerboard contains each combination of the two antibiotics, and the test tube with the highest concentration of each antibiotic in the opposite corner.
  • In order to quantify the interaction between the tested antibiotics (FIC index), use the following formula to calculate:
    A /MIC A + B/MIC B = FIC A + FIC B= FIC Index
    Where A and B are the MICs of each antibiotic combined (in one well), and MIC A and MIC B are the MICs of each drug, respectively.
  • Then, the FIC Index value is used to classify and judge the interaction of the two tested antibiotics. When the FIC value is less than 0.5, the two antibiotics have a synergistic effect; when FIC> 4, the two antibiotics are antagonistic; when the FIC is 0.5-4, the two are shown as additive or indifference.

Our Antimicrobial Synergy Testing Services

  • Standard bacteria culturing
  • Antimicrobial synergy testing
  • Statistics analysis of experimental results

Our Advantages

  • Ensure high efficiency and quality for antimicrobial synergy detection services
  • Competitive price in the market of antimicrobial synergy testing services
  • Ensure 24/7 online service
  • Timely result feedback

We combine infection and analytical expertise to provide our clients with the most powerful portfolio of antiviral and antimicrobial in vitro testing services. Facing an increasing demand for new antiviral and antimicrobial compounds for the treatment of infectious diseases, Creative Diagnostics can test these compounds in vitro to determine their potential efficacy in vivo models.


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We Are Providing Our Clients Antiviral & Antibacterial Services

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Antiviral Services - Creative Diagnostics

Creative Diagnostics provides expertise in antiviral (or antibacterial) assays and cell-based antiviral (or antibacterial) compound screening to other biotechnology and pharmaceutical companies.

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