Synergy assessed by checkerboard. A critical analysis

Diagn Microbiol Infect Dis. May-Jun 1993;16(4):343-9. doi: 10.1016/0732-8893(93)90087-n.


The checkerboard dilution test is widely used for evaluation of in vitro synergy for multiple drugs, although problems in performance, standardization, and interpretation have been noted. A major problem inherent in this commonly used method is the use of twofold dilutions for the antibiotic concentrations. We evaluated an alternative method proposed by Horrevorts and colleagues that preserved the twofold dilution scheme. Giant checkerboards were constructed from a series of component checkerboards using rifampin and minocycline against Staphylococcus aureus. We found that this method improved the stability of the fractional inhibitory concentration (FIC) indices, but required substantially more labor and generated other problems. FIC interpretation and calculation remained compromised by the twofold dilution scheme. We have analyzed the theoretical basis of the checkerboard and its FIC calculation and conclude that the twofold dilution with its exponential increase in dilutions makes this method of synergy evaluation inherently unstable. The principle of examining growth at multiple dilutions of combined antibiotics is valid for assessment of synergy, but newer methods need to be devised.

MeSH terms

  • Drug Synergism*
  • Evaluation Studies as Topic
  • Indicator Dilution Techniques*
  • Microbial Sensitivity Tests
  • Minocycline / pharmacology
  • Rifampin / pharmacology
  • Staphylococcus aureus / drug effects*
  • Staphylococcus aureus / growth & development


  • Minocycline
  • Rifampin