The Epsilometer Test (E-test) is a quantitative antimicrobial susceptibility testing method used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics against microorganisms.
It is also known as the exponential gradient method because it uses a continuous exponential gradient of antibiotic concentrations.
The E-test is a cost-effective and reliable technique that provides direct quantification of a microorganism's susceptibility or resistance to antimicrobial agents.
This method combines the principles of antibiotic dilution and agar diffusion, offering greater precision than conventional disk diffusion testing.
The test produces an inhibition ellipse, allowing the MIC to be read directly from the strip where the edge of the inhibition zone intersects the concentration scale.
The Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism after incubation.
The E-test uses a rectangular plastic strip impregnated with a predefined, continuous, and exponential gradient of an antibiotic.
One side of the strip contains the antibiotic concentration gradient, while the opposite side displays a numerical concentration scale, enabling direct MIC determination without additional calculations.
The E-test is widely used in clinical microbiology laboratories for antimicrobial susceptibility testing, particularly when accurate MIC values are required for patient management or research.
Objectives of the E-test
To determine the Minimum Inhibitory Concentration (MIC) of an antimicrobial agent against a specific microorganism.
To assess the antimicrobial susceptibility of fastidious, slow-growing, or nutritionally demanding microorganisms that may not be suitable for routine susceptibility testing methods.
To detect and confirm low-level antimicrobial resistance associated with specific resistance phenotypes.
To provide an accurate and quantitative MIC value that helps clinicians select the most effective antimicrobial therapy.
To support clinical decision-making and antimicrobial stewardship by guiding appropriate antibiotic selection and dosage.
Principle of the E-test
The E-test is based on the combined principles of agar diffusion and antibiotic dilution, allowing the direct determination of the Minimum Inhibitory Concentration (MIC) of an antimicrobial agent.
A standardized bacterial suspension is uniformly inoculated onto the surface of a Mueller–Hinton agar (MHA) plate using a sterile cotton swab to create a confluent lawn of bacterial growth.
An E-test strip, containing a predefined continuous exponential gradient of antibiotic concentrations, is carefully placed on the inoculated agar surface.
Once the strip is applied, the antibiotic immediately begins to diffuse from the plastic strip into the agar, creating a stable concentration gradient around the strip.
During incubation, the bacteria grow on the agar except in areas where the antibiotic concentration is sufficient to inhibit growth.
This results in the formation of a symmetrical elliptical zone of inhibition surrounding the E-test strip.
The Minimum Inhibitory Concentration (MIC) is determined by reading the value on the strip's numerical scale at the point where the edge of the inhibition ellipse intersects the strip.
The MIC is expressed in micrograms per milliliter (µg/mL) and represents the lowest concentration of the antimicrobial agent that inhibits visible bacterial growth.
Procedure of the E-test
1. Inoculum Preparation
Remove the E-test strips from the freezer (−20°C) at least 30 minutes before use and allow them to reach room temperature.
Select 3–4 well-isolated colonies of the test microorganism from a fresh culture.
Emulsify the colonies in a tube containing sterile normal saline to prepare a bacterial suspension.
Adjust the turbidity of the suspension to match the 0.5 McFarland standard, ensuring a standardized inoculum for accurate MIC determination.
2. Inoculation of Mueller–Hinton Agar (MHA)
Dip a sterile cotton swab into the standardized bacterial suspension.
Rotate the swab against the inside wall of the tube above the liquid level to remove excess inoculum.
Evenly streak the swab over the entire surface of a Mueller–Hinton agar (MHA) plate to produce a uniform bacterial lawn.
Rotate the agar plate approximately 60° and repeat the streaking process two more times to ensure complete and even coverage.
Finally, run the swab around the rim of the agar plate.
Leave the inoculated plate with the lid closed for 3–5 minutes (but no longer than 15 minutes) to allow excess surface moisture to be absorbed before applying the E-test strip.
3. Application of the E-test Strip
Open the E-test package using aseptic technique.
Using sterile forceps or an E-test applicator, carefully remove one strip from the package.
Place the strip gently onto the agar surface with the antibiotic concentration scale facing upward and fully in contact with the agar.
Position the strip so that the "E" end is near the edge of the plate, ensuring it lies flat without air bubbles or movement.
4. Incubation
Invert the inoculated plate and incubate it at 35–37°C for 18–24 hours under appropriate atmospheric conditions for the test organism.
After incubation, observe the elliptical zone of inhibition around the strip and record the Minimum Inhibitory Concentration (MIC) by reading the value where the edge of the inhibition ellipse intersects the strip's numerical scale.
Result and Interpretation of the E-test
After incubation, examine the agar plate from the top (lid side) under adequate lighting.
A symmetrical elliptical zone of inhibition forms around the E-test strip if the microorganism is susceptible to the antibiotic.
Determine the Minimum Inhibitory Concentration (MIC) by identifying the point where the edge of the inhibition ellipse intersects the numerical scale on the E-test strip.
Read the MIC at the point of complete inhibition of visible bacterial growth. Ignore isolated colonies or faint hazes within the inhibition zone unless specific reading guidelines for the organism or antibiotic indicate otherwise.
If the intersection falls between two concentration values, record the higher (next highest) MIC value.
Express the MIC in micrograms per milliliter (µg/mL).
Compare the obtained MIC value with the Clinical and Laboratory Standards Institute (CLSI) or other recognized breakpoint guidelines to interpret the antimicrobial susceptibility of the isolate.
Based on the established breakpoint values, the microorganism is classified as:
Susceptible (S): The antimicrobial is expected to inhibit the organism at the recommended therapeutic dose.
Intermediate (I): The antimicrobial may be effective under specific conditions, such as higher drug exposure or when the drug concentrates at the site of infection.
Resistant (R): The organism is not inhibited by clinically achievable concentrations of the antimicrobial, making treatment with that antibiotic unlikely to be effective.
Precautions
Follow strict aseptic techniques throughout the procedure to prevent contamination and ensure reliable results.
Observe appropriate biosafety precautions when handling bacterial cultures and clinical specimens.
Perform the E-test exactly according to the manufacturer's instructions and standard laboratory protocols.
Ensure the bacterial inoculum is standardized to the 0.5 McFarland standard, as an incorrect inoculum density can affect the MIC result.
Place the E-test strip carefully onto the agar surface using sterile forceps or an applicator, ensuring it lies flat with complete contact and without trapping air bubbles.
Avoid touching the edge of the agar plate or disturbing the strip after placement, as movement can alter the antibiotic gradient and affect the results.
Allow the inoculated agar surface to dry briefly (3–5 minutes) before applying the strip to prevent uneven diffusion of the antibiotic.
If multiple E-test strips are placed on the same agar plate, ensure they are adequately spaced and do not touch or overlap, as overlapping inhibition zones can interfere with accurate MIC interpretation.
Incubate the plates under the recommended temperature, atmosphere, and incubation time for the test organism.
Interpret the results only when a well-defined, symmetrical inhibition ellipse is visible. Poor or irregular inhibition zones may produce inaccurate MIC values.
Always interpret MIC values using the latest CLSI or EUCAST breakpoint guidelines for the specific microorganism and antimicrobial agent.
Advantages of the E-test
Simple and easy to perform, requiring minimal technical training and laboratory expertise.
Provides a direct and accurate determination of the Minimum Inhibitory Concentration (MIC) for antimicrobial agents.
Combines the advantages of agar diffusion and antibiotic dilution methods in a single test.
Less time-consuming and more convenient than conventional broth dilution methods for MIC determination.
Suitable for testing a wide range of antibiotics, antifungal agents, and microorganisms, including fastidious and slow-growing organisms.
Allows easy visualization of contamination or mixed bacterial growth because the inhibition ellipse can be directly observed.
Useful for detecting and confirming specific antimicrobial resistance phenotypes, including low-level resistance.
Helps identify emerging or novel resistance mechanisms that may not be detected by routine susceptibility tests.
Particularly valuable for detecting reduced susceptibility or resistance to Amphotericin B in certain fungal isolates.
Can be used to evaluate the synergistic effects of antimicrobial combination therapies by placing two E-test strips in specific configurations.
Produces quantitative, reproducible, and clinically relevant MIC values, which aid in selecting appropriate antimicrobial therapy and monitoring resistance patterns.
Limitations of the E-test
The E-test is more expensive than conventional disk diffusion methods, especially when testing multiple antimicrobial agents.
It may not be suitable for all microorganisms, as standardized testing protocols and interpretive criteria are unavailable for some species. For example, it is not routinely recommended for Cryptococcus neoformans in certain testing applications.
Accurate MIC determination depends on proper inoculum preparation, strip placement, incubation conditions, and correct interpretation of the inhibition ellipse.
Reading the MIC can be challenging when the inhibition ellipse is irregular, exhibits trailing growth, or contains microcolonies within the inhibition zone.
Results may vary depending on the agar medium, incubation conditions, and the antimicrobial agent being tested.
When evaluating combination antimicrobial therapy, interpreting interactions between two agents can be difficult because it may be challenging to distinguish synergistic, additive, indifferent, or antagonistic effects.
The E-test is generally less suitable for high-throughput susceptibility testing compared with automated antimicrobial susceptibility testing (AST) systems.
Proper storage of E-test strips (usually at −20°C or according to the manufacturer's recommendations) is essential, as improper storage can reduce antibiotic stability and affect test accuracy.
References
Bailey, W. R., Scott, E. G., & Tille, P. M. (2014). Bailey & Scott's Diagnostic Microbiology (13th ed.). Elsevier Mosby.
Joyce, L. F., Downes, J., Stockman, K., & Andrew, J. H. (1992). Comparison of five methods, including the PDM Epsilometer Test (E-test), for antimicrobial susceptibility testing of Pseudomonas aeruginosa. Journal of Clinical Microbiology, 30(10), 2709–2713.
JoVE Science Education. Antibiotic Susceptibility Testing: Epsilometer Tests to Determine the Minimum Inhibitory Concentration (MIC). Available at: https://www.jove.com/science-education/10512/antibiotic-susceptibility-testing-epsilometer-tests-to-determine-mic
Sader, H. S., & Pignatari, A. C. C. (1994). E-test: A novel technique for antimicrobial susceptibility testing. São Paulo Medical Journal, 112(4).
Microbe Online. E-test (Epsilometer Test): Principle, Purpose, Procedure, Results and Interpretation. Available at: https://microbeonline.com/e-test-epsilometer-test-principle-purpose-procedure-results-and-interpretations/
Microbiology Notes. E-test (Epsilometer Test): Principle, Purpose, Procedure, Result Interpretation, Precautions, Advantages and Disadvantages. Available at: https://microbiologynotes.com/e-epsilometer-test-principle-purpose-procedure-result-interpretation-with-precaution-advantages-and-disadvantages/
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Becton, Dickinson and Company (BD). ETEST Technical Information. Available at: http://legacy.bd.com/ds/technicalCenter/inserts/L007393(11)(0706).pdf
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DermNet NZ. Laboratory Tests for Bacterial Infections. Available at: https://dermnetnz.org/topics/laboratory-tests-for-bacterial-infections/
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