Etest for MIC Determination: How to Read and Interpret Results Accurately

The Etest (bioMérieux) is a quantitative antimicrobial susceptibility testing method that uses a predefined, continuous gradient of an antimicrobial agent immobilized on a plastic strip to determine the minimum inhibitory concentration (MIC) of a bacterial isolate. The MIC is read directly from the strip where the elliptical zone of growth inhibition intersects the graded scale. This method is particularly useful when a precise MIC value is needed for clinical decision-making, resistance surveillance, or epidemiological studies, and it offers a practical alternative to broth microdilution for laboratories without automated systems. Accurate reading and interpretation require careful attention to the inhibition ellipse shape, the strip scale, and common artifacts that can lead to misclassification.

At a Glance

Scientific Principle of the Etest

The Etest is a gradient diffusion method that combines the principle of dilution (continuous antimicrobial gradient) with diffusion (radial diffusion from the strip into the agar). A predefined, exponential concentration gradient of the antimicrobial agent is immobilized on one side of a thin, inert plastic strip. When the strip is placed on an agar plate inoculated with a standardized bacterial suspension, the antimicrobial agent diffuses immediately into the surrounding agar, establishing a continuous concentration gradient that decreases with distance from the strip.

After incubation, bacterial growth is visible as a lawn on the agar surface, except in a zone around the strip where the antimicrobial concentration is sufficient to inhibit growth. This zone of inhibition is elliptical, with the long axis parallel to the strip. The MIC is read at the point where the margin of the inhibition ellipse intersects the numerical scale printed on the strip. The scale is calibrated in doubling dilution steps (e.g., 0.016, 0.032, 0.064, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 µg/mL), corresponding to standard MIC dilutions.

The method is based on the same principle as broth microdilution but uses a solid medium (Mueller-Hinton agar) and a single strip per antimicrobial agent. The continuous gradient provides a finer resolution than the discrete twofold dilutions of broth microdilution, potentially allowing detection of subtle MIC changes that might be missed by the twofold dilution series [1].

Materials and Instrumentation Choices

Agar Medium

The standard medium for Etest is Mueller-Hinton agar (MHA). For fastidious organisms, supplemented media are required:

• Streptococcus pneumoniae: MHA with 5% defibrinated sheep blood (MH-F)
• Haemophilus influenzae: Haemophilus Test Medium (HTM)
• Neisseria gonorrhoeae: GC agar base with 1% defined growth supplement
• Anaerobic bacteria: Brucella blood agar supplemented with hemin and vitamin K1

The choice of agar brand and lot can affect MIC results. A multicenter study found that Etest benzylpenicillin and ampicillin strips on Oxoid plates showed lower essential agreement with broth microdilution (58.3% and 65.8%, respectively) compared to BD BBL plates (84.2% for ampicillin) when testing Streptococcus pneumoniae [2]. This demonstrates that agar brand can significantly influence results, and laboratories should validate their specific agar–strip combinations or follow manufacturer recommendations.

Inoculum Preparation

The inoculum should be prepared from an 18–24 hour pure culture. The standard turbidity is 0.5 McFarland standard (approximately 1–2 × 10⁸ CFU/mL for most bacteria). For some fastidious organisms, a higher inoculum (1.0 McFarland) may be recommended. The inoculum should be used within 15–30 minutes of preparation to avoid changes in bacterial viability.

Strip Handling and Storage

Etest strips are stored at -20°C until use. Before opening the foil pouch, allow the strips to reach room temperature (approximately 30 minutes) to prevent condensation. Strips should be handled by the upper end (marked with the antimicrobial abbreviation) using sterile forceps or a dispenser. Once removed from the pouch, strips must be used within the same day.

Incubation Conditions

Standard incubation is 35 ± 2°C in ambient air for 16–24 hours. For fastidious organisms, 5% CO₂ may be required. Anaerobic organisms require anaerobic incubation for 48 hours. The incubation time and atmosphere must be consistent with the specific organism–antimicrobial combination being tested.

Controls and Quality Assurance

Reference Strains

Quality control (QC) strains with known MIC ranges must be tested:

• Escherichia coli ATCC 25922 (for Gram-negative antimicrobials)
• Staphylococcus aureus ATCC 29213 (for Gram-positive antimicrobials)
• Pseudomonas aeruginosa ATCC 27853 (for antipseudomonal agents)
• Enterococcus faecalis ATCC 29212 (for Gram-positive agents)
• Streptococcus pneumoniae ATCC 49619 (for fastidious organism testing)

QC should be performed:

• Weekly for routine testing
• With each new lot of strips or agar
• When any change in testing conditions occurs
• When training new personnel

The acceptable MIC range for each QC strain–antimicrobial combination is provided by the manufacturer and should be within ±1 twofold dilution of the expected value. If QC results fall outside the acceptable range, the test is invalid, and corrective action must be taken (e.g., check inoculum, medium, incubation conditions, strip integrity).

Internal Controls

Each Etest strip includes an internal control: the strip itself contains a predefined gradient, and the scale is calibrated by the manufacturer. However, the user must verify that:

• The strip is not expired or damaged
• The agar depth is consistent (4.0 ± 0.5 mm for MHA)
• The inoculum is correct (confluent but not heavy growth)
• Incubation conditions are appropriate

Conceptual Workflow for Etest Reading

Step 1: Visual Inspection of the Plate

Before reading any strip, examine the entire plate for:

• Adequate growth (confluent lawn without gaps)
• Contamination (colonies with different morphology)
• Proper strip adhesion (strip should be in full contact with agar)
• Absence of condensation droplets that could distort the inhibition zone

Step 2: Identify the Inhibition Ellipse

The inhibition zone should be elliptical, with the long axis centered on the strip. The ellipse margin is defined as the point where there is an obvious reduction in growth density. This is typically a sharp transition from visible growth to no growth.

Step 3: Read the MIC Value

Read the MIC at the point where the ellipse margin intersects the strip scale. The reading point is:

• For most organisms: the first point of complete inhibition
• For trailing growth (e.g., with some β-lactams and Gram-negative organisms): read at 80–90% inhibition (see Troubleshooting section)
• For double zones: read at the inner zone margin (see Troubleshooting section)

The MIC is read to the nearest scale marking. If the intersection falls between two markings, round up to the next higher dilution (more conservative, indicating higher MIC).

Step 4: Record the Result

Record the MIC value in µg/mL. Also note:

• Any artifacts or unusual features
• The reading point (e.g., "read at inner zone" or "trailing growth noted")
• QC results for the batch

Quality Checks During Reading

Ellipse Symmetry

The inhibition ellipse should be symmetrical around the strip. Asymmetry may indicate:

• Uneven agar depth
• Uneven strip placement
• Contamination on one side of the strip
• Inoculum density variation across the plate

Zone Clarity

The margin should be clear and well-defined. Hazy or indistinct margins may indicate:

• Suboptimal incubation time
• Inoculum too heavy or too light
• Antimicrobial degradation
• Heteroresistance in the population

Microcolonies Within the Ellipse

Small colonies within the inhibition zone indicate the presence of a resistant subpopulation. This is particularly important for detecting heteroresistance, which has been documented in vancomycin-resistant Enterococcus faecium where within-patient MIC increases were detected using Etest [1]. When microcolonies are present:

• Read the MIC at the point where the ellipse margin would be if the microcolonies were absent (i.e., ignore the microcolonies for the primary MIC)
• Report the presence of microcolonies separately
• Consider repeating the test or performing a population analysis profile

Result Interpretation

Clinical Categorization

Once the MIC is determined, it must be interpreted using clinical breakpoints from CLSI (Clinical and Laboratory Standards Institute) or EUCAST (European Committee on Antimicrobial Susceptibility Testing). The MIC is categorized as:

• Susceptible (S): MIC ≤ susceptible breakpoint
• Intermediate (I): MIC between susceptible and resistant breakpoints
• Resistant (R): MIC ≥ resistant breakpoint

The choice of breakpoint organization depends on local laboratory policy and regulatory requirements. EUCAST breakpoints are increasingly used in Europe, while CLSI breakpoints are standard in the United States and many other regions.

Comparison with Broth Microdilution

Etest MICs are generally within ±1 twofold dilution of broth microdilution MICs for most organism–antimicrobial combinations. However, discrepancies can occur. A multicenter evaluation of Streptococcus pneumoniae found that Etest benzylpenicillin and ampicillin on certain agar brands showed essential agreement below 90% with broth microdilution [2]. This underscores the importance of:

• Using validated agar–strip combinations
• Confirming borderline results with a reference method
• Being cautious when the Etest MIC is 1–2 dilutions below the clinical breakpoint

Reporting

Report the MIC value and the clinical category. For example:

• "Penicillin MIC = 0.064 µg/mL (Susceptible)"
• "Cefotaxime MIC = 2 µg/mL (Intermediate)"

If the MIC falls at the breakpoint, repeat the test. If the repeat result is the same, report as "Intermediate" or "Susceptible at increased exposure" depending on the breakpoint system.

Troubleshooting Common Artifacts

Limitations of the Etest Method

Method-Specific Limitations

1. Agar brand dependency: As demonstrated with Streptococcus pneumoniae, Etest results can vary significantly between agar brands [2]. Laboratories must validate their specific agar–strip combinations.

2. Not all combinations validated: Etest strips are not available for all antimicrobial agents, and not all organism–antimicrobial combinations have been validated by the manufacturer.

3. Reading subjectivity: The interpretation of the ellipse margin can be subjective, particularly with trailing growth or double zones. This can lead to inter-reader variability.

4. Cost: Etest strips are more expensive per test than disk diffusion and comparable to broth microdilution for single agents.

5. Throughput: Only one antimicrobial agent can be tested per strip, making it less efficient than automated systems for panels of multiple agents.

Comparison with Other Methods

• Versus broth microdilution: Etest generally shows good correlation but may over- or underestimate MIC for certain combinations [2]. Broth microdilution remains the reference method.
• Versus disk diffusion: Etest provides quantitative MIC values, while disk diffusion provides qualitative (S/I/R) results. Etest is more expensive but gives more precise data.
• Versus automated systems (Vitek 2, BD Phoenix): Automated systems can test multiple agents simultaneously and include expert systems for result validation [3]. However, they may have limitations for certain organism–antimicrobial combinations.

Interpretation Challenges

• Heteroresistance: The presence of a resistant subpopulation can complicate reading. This is clinically relevant, as seen in daptomycin resistance evolution in Enterococcus faecium where within-patient MIC increases were detected [1].
• Polymyxin testing: Polymyxin susceptibility testing by Etest is particularly challenging and may not accurately predict resistance. Machine learning approaches using genomic data have been explored as alternatives [4].
• Borderline results: MICs near the clinical breakpoint require confirmation by a reference method.

Documentation and Reporting

Laboratory Records

Document the following for each Etest:

• Organism identification and source
• Antimicrobial agent tested
• Agar medium and lot number
• Inoculum preparation method and turbidity
• Incubation conditions (temperature, atmosphere, time)
• MIC value and reading point
• Any artifacts or unusual features
• QC results for the batch
• Technician initials and date

Clinical Reporting

For clinical isolates, report:

• MIC value (in µg/mL)
• Clinical category (S, I, R) using the appropriate breakpoint system
• Any comments about unusual features (e.g., "heteroresistance detected")

Research Documentation

For research studies, additional documentation may include:

• Photographs of plates with strips
• Digital images of inhibition ellipses
• Raw MIC data for statistical analysis
• Comparison with reference method results

Biosafety Considerations

Routine BSL-1 Teaching Laboratory

For teaching laboratories using non-pathogenic organisms (e.g., E. coli ATCC 25922, S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212), standard BSL-1 practices apply [5]:

• Standard microbiological practices (hand washing, no eating/drinking)
• Decontamination of work surfaces before and after use
• Proper waste disposal (autoclave all contaminated materials)
• Use of personal protective equipment (lab coat, gloves)

BSL-2 Considerations

When working with clinical isolates or potentially pathogenic organisms (e.g., Streptococcus pneumoniae, Pseudomonas aeruginosa), BSL-2 practices are required [5]:

• All procedures performed in a biological safety cabinet (BSC)
• Restricted access to the laboratory
• Sharps precautions
• Enhanced personal protective equipment (face protection if splashing risk)
• Specific decontamination protocols

Recombinant or Synthetic Nucleic Acid Work

If the Etest is used in research involving recombinant or synthetic nucleic acid molecules (e.g., testing transformed strains), the work must comply with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules [6]. This includes:

• Institutional Biosafety Committee (IBC) approval
• Appropriate containment level based on risk assessment
• Documentation of the recombinant construct and host organism

Frequently Asked Questions

1. How do I read an Etest when there is a double zone of inhibition?

A double zone appears as an inner clear zone surrounded by a hazy outer zone. This typically indicates heteroresistance, where a subpopulation of bacteria has reduced susceptibility. Read the MIC at the inner zone margin (the clear zone), as this represents the true inhibition point for the majority population. Document the presence of the double zone and consider repeating the test or performing a population analysis profile to characterize the resistant subpopulation.

2. What should I do if the inhibition ellipse margin falls between two scale markings?

When the intersection point falls between two markings on the Etest strip scale, round up to the next higher dilution. This is a conservative approach that ensures the reported MIC is not lower than the actual value. For example, if the intersection is between 0.25 and 0.5 µg/mL, report the MIC as 0.5 µg/mL. This practice aligns with the standard approach for broth microdilution, where the MIC is read as the lowest concentration that inhibits visible growth.

3. Can I use Etest strips on agar plates that have been stored for several days?

Etest strips should be placed on freshly prepared agar plates (within 7 days of preparation for MHA stored at 2–8°C). Older plates may have reduced moisture content, which can affect antimicrobial diffusion and lead to inaccurate MIC results. Additionally, plates should be dried before use to remove surface moisture, as excess moisture can cause the antimicrobial to diffuse too rapidly, resulting in a falsely low MIC. Dry plates in a laminar flow hood or incubator with lids slightly ajar for 10–30 minutes before inoculation.

4. How do I interpret Etest results for polymyxins (colistin and polymyxin B)?

Polymyxin susceptibility testing by Etest is notoriously difficult and often unreliable. Polymyxins are large, cationic molecules that diffuse poorly in agar, leading to inconsistent results. Broth microdilution is the recommended reference method for polymyxin MIC determination. If Etest is used, results should be interpreted with extreme caution, and any susceptible result should be confirmed by broth microdilution. Genomic approaches, including machine learning analysis of whole-genome sequencing data, are being explored as alternative methods for predicting polymyxin resistance [4].

References and Further Reading

1. Kinnear CL, Patel TS, Young CL, et al. Impact of an Antimicrobial Stewardship Intervention on Within- and Between-Patient Daptomycin Resistance Evolution in Vancomycin-Resistant Enterococcus faecium. 2019. https://pubmed.ncbi.nlm.nih.gov/30718245/

   • Demonstrates use of Etest for detecting within-patient MIC changes and resistance evolution.

2. Martens S, Cuypers L, Bélik F, et al. Multicenter comparison of Etest, Vitek2 and BD Phoenix to broth microdilution for beta-lactam susceptibility testing of Streptococcus pneumoniae. 2024. https://pubmed.ncbi.nlm.nih.gov/38801483/

   • Provides evidence of agar brand effects on Etest accuracy and essential agreement with broth microdilution.

3. Winstanley T, Courvalin P. Expert systems in clinical microbiology. 2011. https://pubmed.ncbi.nlm.nih.gov/21734247/

   • Reviews automated systems and interpretive reading, including context for Etest use in clinical laboratories.

4. Macesic N, Bear Don't Walk OJ, Pe'er I, et al. Predicting Phenotypic Polymyxin Resistance in Klebsiella pneumoniae through Machine Learning Analysis of Genomic Data. 2020. https://pubmed.ncbi.nlm.nih.gov/32457240/

   • Discusses challenges of phenotypic polymyxin testing and alternative genomic approaches.

5. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. 2020. https://www.cdc.gov/labs/bmbl/index.html

   • Authoritative source for biosafety practices in microbiological laboratories.

6. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules/

   • Framework for biosafety in recombinant nucleic acid research.

7. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. https://www.ncbi.nlm.nih.gov/books/

   • Searchable collection of biomedical methods references and protocols.

Related Articles

• How to Read and Interpret an Etest Strip for MIC Determination
• How to Perform an Etest for MIC Determination: Protocol and Interpretation
• How to Interpret Kirby-Bauer Disk Diffusion Results Using CLSI Breakpoints
• Minimum Inhibitory Concentration (MIC) Determination by Broth Microdilution: A Practical Protocol
• How to Perform an Antibiotic Susceptibility Test Using the Etest Method
• Understanding CLSI Breakpoints for Disk Diffusion and MIC Interpretation