How to Perform an Antibiotic Susceptibility Test Using the Etest Method

The Etest (also known as gradient diffusion strip method) is a quantitative antimicrobial susceptibility testing technique that uses a plastic strip impregnated with a predefined continuous concentration gradient of an antimicrobial agent to determine the minimum inhibitory concentration (MIC) of a bacterial isolate. This method is particularly useful when a precise MIC value is needed for clinical decision-making, epidemiological surveillance, or research applications, as it provides a direct numerical readout rather than the categorical susceptible/intermediate/resistant result obtained from disk diffusion. The Etest bridges the gap between the simplicity of disk diffusion and the quantitative precision of broth microdilution, making it a practical choice for laboratories that require MIC data without the complexity of preparing serial dilutions.

At a Glance

Scientific Principle of the Gradient Diffusion Method

The Etest method relies on the establishment of a stable, continuous concentration gradient of an antimicrobial agent along the length of a plastic strip. The strip is approximately 5 mm wide and 60 mm long, with the antimicrobial concentration decreasing from one end to the other. When placed on an agar plate inoculated with a standardized bacterial suspension, the antimicrobial agent diffuses radially into the agar, creating a concentration gradient that mirrors the gradient on the strip.

Bacterial growth occurs in regions where the antimicrobial concentration is below the MIC, while growth is inhibited where the concentration exceeds the MIC. After incubation, an elliptical zone of inhibition forms around the strip, with the intersection of the ellipse edge and the strip scale indicating the MIC value. The shape of this ellipse is critical: a sharp, well-defined edge at the intersection point provides the most accurate reading, while fuzzy or irregular edges may indicate technical issues or organism-specific phenomena such as trailing growth or heteroresistance.

The gradient diffusion method has been validated against reference broth microdilution for numerous organism-antimicrobial combinations. For example, a multicenter evaluation of the Etest for aztreonam-avibactam against Enterobacterales demonstrated essential agreement of 95.7% and categorical agreement of 98.0% when using FDA interpretive breakpoints [3]. However, performance can vary by organism and antimicrobial agent; studies on Burkholderia cepacia complex have shown that gradient diffusion methods may produce lower MICs compared to reference methods, leading to higher rates of very major errors (false susceptibility) [1].

Materials and Instrumentation Choices

Agar Medium Selection

Mueller-Hinton agar (MHA) is the standard medium for Etest procedures, as recommended by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The medium must be prepared according to the manufacturer's instructions, with careful attention to pH (7.2–7.4 at 25°C), depth (4 mm ± 0.5 mm in a 100 mm plate), and sterility. Variations in medium composition can affect MIC results; for instance, studies have shown that MICs for streptomycin against E. coli are consistently higher on tryptic soy agar compared to Mueller-Hinton agar [4]. For fastidious organisms, supplemented media such as Mueller-Hinton agar with 5% sheep blood or Haemophilus test medium may be required.

Inoculum Preparation

The bacterial inoculum must be standardized to achieve a confluent lawn of growth. The standard method involves suspending colonies from an 18–24 hour pure culture in sterile saline or Mueller-Hinton broth to a turbidity equivalent to a 0.5 McFarland standard (approximately 1–2 × 10⁸ CFU/mL for most organisms). Turbidity should be measured using a calibrated nephelometer or photometric device; visual comparison to McFarland standards is acceptable for teaching laboratories but introduces variability. The inoculum should be used within 15–30 minutes of preparation to maintain viability.

Etest Strips

Etest strips are commercially available from several manufacturers (e.g., bioMérieux, Liofilchem, Oxoid) and cover a wide range of antimicrobial agents. Each strip is labeled with a two-letter code identifying the antimicrobial agent, a concentration scale in µg/mL, and an expiration date. Strips must be stored at -20°C or -80°C as recommended by the manufacturer and allowed to reach room temperature in their sealed container before opening to prevent condensation. Once opened, strips should be used within the timeframe specified in the package insert, typically within one week if stored in a desiccated container at 2–8°C.

Quality Control Strains

Quality control (QC) strains with known MIC ranges for each antimicrobial agent are essential for validating test performance. Commonly used QC strains include Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212. These strains should be tested in parallel with clinical isolates on each day of testing, or at minimum weekly, depending on laboratory protocol. QC results must fall within published acceptable ranges; if they do not, all results from that testing session are invalid and must be repeated.

Controls and Quality Assurance

Internal Controls

Each Etest run must include a growth control (no antimicrobial strip) to confirm that the medium supports adequate growth and that the inoculum is viable. Additionally, a sterility control (uninoculated plate) should be incubated to verify that the medium and reagents are free from contamination.

External Quality Assessment

Participation in external quality assessment (EQA) programs, such as those offered by the College of American Pathologists (CAP) or national reference laboratories, provides independent validation of testing accuracy. EQA samples should be tested using the same protocol as routine isolates, and results should be reviewed for concordance with expected values.

Documentation

All QC results, including MIC values, acceptable ranges, and any corrective actions taken, must be documented in a laboratory quality manual or electronic system. This documentation is critical for accreditation audits and for identifying trends that may indicate systematic shifts in test performance. A recent study demonstrated that subtle shifts in daptomycin MIC results over a three-year period were detectable only through systematic review of patient data and QC records, highlighting the importance of ongoing data analysis beyond routine QC checks [5].

Conceptual Workflow

Step 1: Inoculum Preparation and Plate Inoculation

Prepare a bacterial suspension equivalent to a 0.5 McFarland standard from an 18–24 hour pure culture. Within 15 minutes, dip a sterile cotton swab into the suspension, rotate it against the tube wall to remove excess liquid, and streak the entire surface of a Mueller-Hinton agar plate in three directions to ensure confluent growth. Allow the plate to dry for 10–15 minutes at room temperature with the lid slightly ajar.

Step 2: Strip Application

Using sterile forceps or the applicator provided by the manufacturer, apply the Etest strip to the dried agar surface. The strip should be placed with the scale facing upward and the MIC scale oriented toward the center of the plate. Ensure complete contact between the strip and the agar by gently pressing down on the strip with the forceps, avoiding air bubbles. For plates containing multiple strips, arrange them radially with at least 20 mm between strips to prevent overlapping inhibition zones.

Step 3: Incubation

Incubate the plate inverted (agar side up) at 35°C ± 1°C in ambient air for 16–24 hours. For fastidious organisms, incubation conditions may require 5% CO₂ or extended incubation times. Do not stack plates more than three high to ensure uniform temperature distribution.

Step 4: Reading the MIC

After incubation, examine the plate for an elliptical zone of inhibition surrounding the strip. The MIC is read at the point where the edge of the ellipse intersects the strip scale. For most organism-antimicrobial combinations, the MIC is read at the complete inhibition point, ignoring faint growth or microcolonies within the ellipse. However, for certain antimicrobials (e.g., trimethoprim-sulfamethoxazole, fluconazole), reading at 80% inhibition may be recommended; consult the manufacturer's instructions for specific guidance.

Step 5: Interpretation

Compare the MIC value to established clinical breakpoints (CLSI or EUCAST) to determine the categorical interpretation (susceptible, intermediate, or resistant). Document the MIC value, interpretation, and any relevant observations (e.g., trailing growth, skipped wells in broth microdilution correlation).

Quality Checks and Validation

Acceptance Criteria for QC Strains

QC strains must produce MIC values within the published acceptable range for each antimicrobial agent tested. If a QC result falls outside the acceptable range, investigate potential causes:

• Inoculum density: Verify the McFarland standard using a calibrated device.
• Medium quality: Check pH, depth, and sterility of the agar.
• Strip integrity: Confirm that strips were stored correctly and are within expiration.
• Incubation conditions: Verify temperature, atmosphere, and duration.

Reproducibility Testing

For research applications, reproducibility should be assessed by testing a subset of isolates in triplicate on separate days. Acceptable reproducibility is typically defined as MIC values within one twofold dilution for at least 95% of replicates. Interlaboratory reproducibility for gradient diffusion methods can vary; one study reported reproducibility of 76.5–80% for a gradient diffusion strip stacking method, compared to 93.1% for reference broth microdilution [2].

Trending Analysis

Regular review of QC data can identify subtle shifts in test performance that may not be apparent from individual QC results. Plotting MIC values for QC strains over time (e.g., using Levey-Jennings charts) allows detection of drift or bias. If a systematic shift is observed, investigate potential causes such as changes in medium lot number, reagent expiration, or equipment calibration.

Result Interpretation

Reading the Ellipse

The MIC is read at the point where the edge of the inhibition ellipse intersects the strip scale. The ellipse should be smooth and symmetrical; irregularities may indicate technical problems or organism-specific phenomena.

• Sharp ellipse edge: Read at the intersection point.
• Fuzzy or irregular edge: May indicate trailing growth, heteroresistance, or contamination. Repeat the test.
• Double ellipse: May indicate a mixed culture or contamination. Subculture and repeat.
• No ellipse: Possible complete resistance (MIC > highest concentration on strip) or technical failure (e.g., expired strip, incorrect inoculum).

MIC Values Between Scale Markings

When the ellipse edge falls between two markings on the strip scale, round up to the next higher concentration. For example, if the edge falls between 0.5 and 1.0 µg/mL, report the MIC as 1.0 µg/mL.

Off-Scale Results

• MIC > highest concentration: Report as > [highest concentration] (e.g., >256 µg/mL). This indicates resistance.
• MIC < lowest concentration: Report as ≤ [lowest concentration] (e.g., ≤0.016 µg/mL). This indicates susceptibility.

Categorical Interpretation

Apply the appropriate clinical breakpoints (CLSI M100 or EUCAST breakpoint tables) to convert the MIC to a categorical result. Note that breakpoints may differ between organizations and may change over time; always use the most current version.

Troubleshooting

Limitations of the Etest Method

Cost and Throughput

Etest strips are significantly more expensive per test than disk diffusion discs, making the method less suitable for high-volume screening or resource-limited settings. For laboratories processing large numbers of isolates, broth microdilution panels may offer better cost-effectiveness.

Organism-Specific Performance

The accuracy of the Etest varies by organism and antimicrobial agent. For example, studies on Burkholderia cepacia complex have shown that gradient diffusion methods may produce lower MICs compared to reference broth microdilution, leading to higher rates of very major errors (false susceptibility) [1]. Similarly, performance for metallo-beta-lactamase-producing Enterobacterales can be variable, with one study reporting essential agreement ranging from 46.1% to 92.2% depending on the specific method and testing site [2].

Technical Variability

Subtle technical factors can influence Etest results, including inoculum density, agar depth, incubation conditions, and strip storage. A recent study documented a systematic shift in daptomycin MIC results over three years that was undetectable through routine QC but became apparent through analysis of patient data [5]. This highlights the importance of ongoing monitoring and the potential for unrecognized technical variability.

Inability to Detect Heteroresistance

Standard Etest reading at the complete inhibition point may miss heteroresistant subpopulations that grow within the inhibition ellipse. Specialized techniques such as population analysis profiling are required to detect heteroresistance.

Documentation and Reporting

Required Information for Each Test

• Patient or sample identifier
• Organism identification
• Antimicrobial agent tested
• MIC value (in µg/mL)
• Categorical interpretation (S, I, R)
• QC strain results for the testing session
• Date of testing
• Technician initials

Reporting Format

MIC results should be reported as numerical values with the appropriate units (µg/mL). For off-scale results, use the appropriate notation (e.g., >256 or ≤0.016). The categorical interpretation should be clearly indicated, and any comments (e.g., "indeterminate due to trailing growth") should be included.

Record Retention

Maintain all QC records, test results, and corrective action documentation according to laboratory policy and regulatory requirements, typically for a minimum of two years or as specified by accreditation bodies.

Biosafety Considerations

Risk Assessment

The Etest procedure involves handling live bacterial cultures and should be performed in a biological safety cabinet (BSC) when working with organisms of Risk Group 2 or higher. For teaching laboratories using non-pathogenic organisms (e.g., E. coli ATCC 25922, S. aureus ATCC 29213), BSL-1 practices are appropriate, but standard microbiological precautions should still be observed [6].

Personal Protective Equipment

Wear a laboratory coat, gloves, and eye protection when performing the procedure. Change gloves between handling different isolates to prevent cross-contamination.

Waste Disposal

All contaminated materials (plates, swabs, strips) must be disposed of in biohazard waste containers and autoclaved before disposal. Liquid waste should be treated with an appropriate disinfectant (e.g., 10% bleach solution) before disposal.

Spill Management

In the event of a spill, cover the area with absorbent material, apply a disinfectant (e.g., 1% sodium hypochlorite), allow 20 minutes of contact time, and clean up using appropriate PPE. Document the incident according to laboratory policy.

Frequently Asked Questions

1. Can I use the Etest for all bacterial species and antimicrobial agents?

No. The Etest has been validated for specific organism-antimicrobial combinations, and performance can vary. For example, studies on Burkholderia cepacia complex have shown that gradient diffusion methods may produce lower MICs compared to reference methods, leading to higher rates of very major errors [1]. Always consult the manufacturer's instructions and published validation data for your specific organism-antimicrobial combination. For organisms or agents without published validation, consider using a reference method such as broth microdilution.

2. How do I read the MIC when the ellipse edge is fuzzy or irregular?

A fuzzy or irregular ellipse edge may indicate trailing growth, heteroresistance, or contamination. For some antimicrobials (e.g., trimethoprim-sulfamethoxazole, fluconazole), reading at 80% inhibition is recommended by the manufacturer. If the edge remains unclear after following manufacturer guidance, repeat the test with a fresh inoculum and new strip. If the problem persists, consider using an alternative method such as broth microdilution.

3. What should I do if my QC strain result falls outside the acceptable range?

First, do not report any patient or research results from that testing session. Investigate potential causes systematically: check inoculum density using a calibrated device, verify medium pH and depth, confirm strip storage conditions and expiration, and review incubation conditions. Repeat the QC test with fresh reagents. If the problem persists, contact the manufacturer for technical support and consider using a different lot of strips or medium.

4. How does the Etest compare to broth microdilution for accuracy?

The Etest generally shows good essential agreement (within one twofold dilution) with reference broth microdilution for many organism-antimicrobial combinations, with reported essential agreement often exceeding 90% [3]. However, performance can vary, and some studies have reported lower agreement for specific organisms or agents [1,2]. The Etest is generally more convenient than broth microdilution for testing individual isolates but may be less suitable for high-throughput applications or for organisms where performance has not been validated.

References and Further Reading

1. Shier KL, Gonzales G, Perez DAG, Bao JR, Azad KN. Performance of the Vitek 2, Sensititre, and Etest antimicrobial susceptibility testing systems for isolates of Burkholderia cepacia complex. 2026. PubMed ID: 41538785. Link — Evaluates Etest performance against reference methods for a challenging organism.

2. Bhatnagar AS, Harris H, Jacobs E, et al. Comparison of laboratory-developed methods for aztreonam plus ceftazidime-avibactam antimicrobial susceptibility testing for metallo-beta-lactamase-producing Enterobacterales. 2026. PubMed ID: 42171586. Link — Compares gradient diffusion strip methods to reference broth microdilution.

3. Bui TI, Csiki-Fejer E, Prudhomme S, et al. A multicenter performance evaluation of aztreonam-avibactam gradient diffusion susceptibility testing for Enterobacterales. 2026. PubMed ID: 42007819. Link — Large multicenter validation of Etest for a novel antimicrobial combination.

4. Lakes JE, Ferrell JL, Flythe MD. The effect of biochanin A on the antibiotic susceptibilities of Escherichia coli grown under different respiration and metabolic conditions on agar media. 2026. PubMed ID: 41845216. Link — Demonstrates how medium composition and incubation conditions affect Etest MIC results.

5. Zaydman MA, Glaser L, Herman DS, et al. Leveraging patient data to detect systematic shifts in daptomycin susceptibility testing associated with reduced prescribing. 2026. PubMed ID: 41636497. Link — Highlights the importance of ongoing data analysis for detecting subtle shifts in Etest performance.

6. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services, 2020. Link — Authoritative biosafety guidelines for microbiological laboratory practice.

7. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Link — Biosafety framework for recombinant nucleic acid research.

8. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. Link — Searchable collection of authoritative biomedical methods references.

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