About this FIC Calculator Tool
This free online FIC calculator helps microbiologists, researchers, and students calculate the
Fractional Inhibitory Concentration (FIC) index
from
Minimum Inhibitory Concentrations (MIC)
values.
Based on the
checkerboard assay method
, this tool evaluates
antibiotic synergy
,
additivity
,
indifference
, or
antagonism
between two antimicrobial agents.
Designed for speed and clarity, this calculator is ideal for labs and individuals looking to avoid manual calculations or Excel templates.
Etest (Gradient Diffusion Strip) Synergy Testing: Principle, FIC Calculation, and Practical Method
Beyond the broth microdilution checkerboard, the Etest gradient diffusion strip (bioMérieux) offers an agar-based alternative for estimating the Fractional Inhibitory Concentration (FIC) index of an antibiotic combination. Because each strip carries a continuous, pre-defined antibiotic gradient that directly reads out a Minimum Inhibitory Concentration (MIC) at the point where the elliptical inhibition zone intersects the strip scale, two strips can be combined on a single agar plate to approximate a two-drug interaction without running a full dilution series (White et al., 1996, Antimicrob Agents Chemother 40:1914–1918, doi:10.1128/aac.40.8.1914).
1. Principle of Etest Synergy Methods
Three placement geometries are described in the literature, each altering how the two concentration gradients meet on the agar:
- Cross (90-degree) method: the MIC of each antibiotic is first determined individually, then both strips are laid perpendicular to one another so that the two previously determined MIC graduations intersect at a right angle. This is the original configuration validated by White et al. (1996) against checkerboard and time-kill assays.
- Overlapping / sequential method: the first strip is applied and left in place for a defined intermediate incubation period (commonly around 60 minutes), then removed; the second strip is placed directly over the same groove it leaves in the agar, so the second gradient diffuses through agar already exposed to the first drug (Doern, 2014, J Clin Microbiol 52:4124–4128, doi:10.1128/JCM.01121-14).
- Parallel placement / MIC:MIC ratio method: both strips are placed side by side (not crossed), positioned according to the ratio of their individually determined MICs, which some evaluations have found gives the best correlation with the checkerboard among gradient methods (Okoliegbe et al., 2021, Antibiotics 10:967, doi:10.3390/antibiotics10080967).
2. Calculating and Interpreting the FIC via Etest
Whichever geometry is used, the combined MIC of each drug is read directly off its strip at the point where the inhibition ellipse crosses the scale, and the FIC index is calculated exactly as in the checkerboard method:
FICA = MICA (in combination) / MICA (alone)
FICB = MICB (in combination) / MICB (alone)
FICI = FICA + FICB
The result is interpreted using the standard thresholds also used by the calculator on this page: FICI ≤ 0.5 (synergy), 0.5 < FICI ≤ 1 (additive effect), 1 < FICI ≤ 4 (indifference), and FICI > 4 (antagonism).
The visual reading step is where Etest synergy testing differs most from a liquid checkerboard: instead of scoring turbidity in 96-well plates, the operator must locate the exact point on the agar where the two elliptical inhibition zones truncate one another, and read the MIC on the strip's printed scale at that intersection — a step that requires good lighting, a plate held against a dark background, and a consistent eye-level angle to avoid parallax error.
3. Practical Step-by-Step Guide
- Inoculum preparation: prepare a 0.5 McFarland suspension of the test organism and swab it evenly across a Mueller-Hinton agar plate to obtain confluent growth, following standard Etest susceptibility procedure.
- Individual MIC strips: where the protocol requires pre-determined MICs (cross and MIC:MIC ratio methods), run each antibiotic strip alone first to establish the individual MIC values.
- First strip placement: apply the first antibiotic strip at the position dictated by the chosen method (centered for the cross method, offset by the MIC ratio for the parallel method).
- Intermediate incubation or immediate transfer: for the sequential/overlapping method, incubate briefly (≈1 hour) before removing the first strip; for the cross and MIC:MIC ratio methods, proceed directly to placing the second strip.
- Second strip placement: position the second antibiotic strip so that its gradient intersects (cross method) or overlays (sequential method) the first, or sits parallel to it at the calculated ratio distance.
- Incubation: incubate the plate at 35°C for 16–20 hours (or per organism-specific CLSI/EUCAST guidance).
- Reading: read each drug's combined MIC at the point where its elliptical inhibition zone meets the intersection with the other strip, then apply the FIC formula above.
Frequently Asked Questions
What are the advantages and limitations of Etest synergy testing compared with the broth checkerboard?
Etest synergy testing needs only one or two agar plates instead of a full 96-well dilution matrix, is faster to set up, and reads visually rather than by turbidity. Its main limitation is resolution: strip gradients offer fewer discrete concentration steps at the relevant range than a full checkerboard, and agreement between the two methods has been reported anywhere from about 60% to over 90% depending on the drug combination and organism (Okoliegbe et al., 2021; Sopirala et al., 2010, Antimicrob Agents Chemother 54:4678–4683, doi:10.1128/AAC.00497-10).
How should reading artifacts at the strip intersection be managed?
Deformed or "keyhole"-shaped ellipses at the crossing point, double zones, and haze within the intersection are common artifacts; they should be read at the outermost point of complete growth inhibition adjoining the strip, cross-checked against the individual-strip MICs, and repeated if the intersection zone is too irregular to identify a clear endpoint.
How reproducible is Etest synergy testing between operators and laboratories?
When inoculum density, agar depth, and reading criteria are standardized, gradient diffusion methods generally show good reproducibility; a clinical-laboratory feasibility evaluation found routine synergy testing with gradient strips practical to implement outside of a research setting (Avery & Nicolau, 2018, J Antimicrob Chemother 73:2264–2265, doi:10.1093/jac/dky165).
Does the choice of geometry (cross vs. MIC:MIC ratio) change the result?
Yes. Comparative work on multidrug-resistant Pseudomonas aeruginosa found that agreement with the checkerboard varied by geometry and by which interpretive criterion (FICI vs. susceptibility breakpoint index) was applied, with the MIC:MIC ratio method showing the strongest correlation among the gradient approaches tested (Okoliegbe et al., 2021).
Are there antibiotics for which Etest synergy testing is particularly unreliable?
Yes — gradient diffusion methods, including Etest, are known to perform poorly for colistin, where they have produced unacceptably high very major error rates against carbapenem-resistant Enterobacterales and Acinetobacter baumannii compared with reference broth microdilution (Dafopoulou et al., 2015, Antimicrob Agents Chemother 59:4625–4630, doi:10.1128/aac.00868-15). For colistin-containing combinations in particular, Etest synergy results should be interpreted cautiously and, where possible, confirmed with a reference broth method.
Read the full step-by-step comparison of the cross vs. overlay technique, clinical validation data, and molecule-specific reading pitfalls (colistin, beta-lactams) in our Etest Synergy Testing Guide →
