Antimicrobial Synergy Study – Checkerboard Testing

Synergy measurement by checkerboard analysis is used to determine the impact on potency of the combination of antibiotics in comparison to their individual activities. This comparison is then represented as the Fractional Inhibitory Concentration (FIC) index value. The FIC index value takes into account the combination of antibiotics that produces the greatest change from the individual antibiotic’s MIC.

To quantify the interactions between the antibiotics being tested (the FIC index), the following equation is used:

A+B=   FICA +   FICB   =   FIC Index
MICA         MICB

where A and B are the MIC of each antibiotic in combination (in a single well), and MICA and MICB are the MIC of each drug individually.

The FIC Index value is then used to categorize the interaction of the two antibiotics tested.

FIC value
Synergy <0.5
Antagonism >4
Additive or indifference 0.5-4

Synergy: When the combination of compounds result in an FIC value of <0.5, then the combination of the compounds increases the inhibitory activity (decrease in MIC) of one or both compounds than the compounds alone.

Additive or indifference: When the combination of compounds results in an FIC value of 0.5 – 4, the combination has no increase in inhibitory activity or a slight increase in inhibitory activity from the additive effect of both compounds combined.

Antagonism: When the combination of compounds results in an FIC value of >4, the combination of compounds increases the MIC, or lowers the activity of the compounds.

Synergy time-kill kinetic assays can be performed to further characterize the synergy observed in the microdilution assay. The synergy time kill-kinetic assay can determine how the combination of compounds affects the bacteria during the growth cycle by determining the rate of killing of the compounds. This assay can also determine whether the combination is bacteriostatic or bactericidal.

Below is a representation of a checkerboard assay where the synergistic activity of two antibiotics, A and B was determined.

Synergy Checkerboard Assay. Depicted here is a typical synergy checkerboard assay setup. Columns 1 to 11 contain 2-fold serial dilutions of Compound A, and rows A to G contain 2-fold serial dilutions of compound B. Column 12 contains a serial dilution of Compound B alone, while row H contains a serial dilution of Compound A alone. These controls are used to determine the MIC value for each test compound, which in turn are used to calculate the FIC value using the formula listed above, and then assessed for synergism, additive/indifference, or antagonism. In this illustration, “no growth” is represented by white circles, and “growth” is represented by yellow circles. The growth control (well H12) is depicted as a yellow circle with bolded outline. The concentrations of Compounds A and B in well D6 resulted in a synergistic effect (FIC < 0.5), while in well C7 the effect was additive or indifference (FIC = 0.5-4).


Lorian 5th edition, Chapter 9 (2005) Antimicrobial Combinations, in Antibiotics In Laboratory Medicine,  pp. 365-441. Lippincott Williams and Wilkins, Philadelphia, PA.