How to Perform an Etest for MIC Determination: Protocol and Interpretation

The Etest (also known as the epsilometer test or gradient diffusion method) is a quantitative antimicrobial susceptibility testing technique that uses a plastic strip impregnated with a continuous exponential gradient of an antimicrobial agent to determine the minimum inhibitory concentration (MIC) of a bacterial or fungal isolate. This method is useful when a precise MIC value is needed for clinical decision-making, epidemiological surveillance, or research applications, particularly for fastidious organisms, slow-growing pathogens, or when testing combinations of antimicrobial agents. The Etest provides a direct MIC reading in micrograms per milliliter (µg/mL) by observing where the elliptical zone of growth inhibition intersects the graded scale on the strip, offering a practical alternative to broth microdilution for laboratories without automated systems.

At a Glance

Scientific Principle of the Gradient Diffusion Method

The Etest relies on the diffusion of a pre-formed antimicrobial gradient from the plastic strip into the agar medium. The strip contains a dried antimicrobial agent that is immobilized on one side, with concentrations ranging from a low value at one end to a high value at the other end, typically covering a 15-log2 dilution series. When the strip is placed on an inoculated agar plate, the antimicrobial agent diffuses radially into the medium, establishing a continuous concentration gradient perpendicular to the strip. After incubation, bacterial growth is inhibited where the local antimicrobial concentration exceeds the MIC of the test organism, creating a symmetrical elliptical zone of inhibition. The MIC is read at the point where the ellipse intersects the strip scale, which is calibrated to the corresponding concentration.

This method differs from disk diffusion in that it provides a quantitative MIC rather than a qualitative susceptible/intermediate/resistant categorization. Studies have shown that Etest results generally correlate well with reference broth microdilution (BMD), with essential agreement rates often exceeding 90% for many organism-antimicrobial combinations [5]. However, performance can vary depending on the specific antimicrobial agent, organism species, and agar medium used. For example, one multicenter study found that Etest for penicillin and ampicillin against Streptococcus pneumoniae showed lower essential agreement (58.3–84.2%) on certain agar brands compared to BMD, highlighting the importance of medium selection [5].

Materials and Instrumentation Choices

Agar Medium Selection

The choice of agar medium is critical for Etest performance. Mueller-Hinton agar (MHA) is the standard medium for most bacterial isolates, as recommended by CLSI and EUCAST guidelines. However, specific organisms require modifications:

• Fastidious bacteria: Supplement MHA with 5% defibrinated sheep blood for Streptococcus spp., Haemophilus spp., and Neisseria spp.
• Anaerobic bacteria: Use Brucella blood agar supplemented with hemin and vitamin K1.
• Yeasts and fungi: Use RPMI 1640 agar with 2% glucose and MOPS buffer, or MHA supplemented with 2% glucose and 0.5 µg/mL methylene blue for Candida spp.

The depth of the agar should be 4 ± 0.5 mm (approximately 25 mL per 100 mm plate). Plates should be prepared fresh or stored at 2–8°C for no more than 7 days in sealed plastic bags to prevent drying. The pH of the medium should be 7.2–7.4 for MHA, as deviations can affect antimicrobial activity.

Etest Strips

Etest strips are commercially available from several manufacturers, including bioMérieux (Etest) and Liofilchem (MIC Test Strip). Each strip contains a specific antimicrobial agent with a defined concentration range printed on the strip. The strips are packaged in individual foil pouches with desiccant and should be stored at -20°C or as recommended by the manufacturer. Allow strips to reach room temperature (15–25°C) for at least 30 minutes before opening the pouch to prevent condensation.

Inoculum Preparation

Prepare a bacterial suspension equivalent to a 0.5 McFarland standard (approximately 1.5 × 10⁸ CFU/mL for bacteria). For yeasts, use a 0.5 McFarland standard (approximately 1–5 × 10⁶ CFU/mL). The suspension should be prepared in sterile saline or broth, and the turbidity should be verified using a nephelometer or photometric device. Visual comparison to a McFarland standard is acceptable for routine use but less precise.

Inoculation and Application

Use a sterile cotton swab to inoculate the entire surface of the agar plate by streaking in three directions to ensure confluent growth. Allow the plate surface to dry for 10–15 minutes before applying Etest strips. Apply strips using sterile forceps or the applicator provided by the manufacturer, pressing gently to ensure complete contact with the agar surface. For multiple strips on one plate, place them radially with the high-concentration end toward the edge of the plate, maintaining at least 15–20 mm between strips to prevent overlapping inhibition zones.

Controls

Quality control is essential for valid Etest results. Include the following controls with each batch of tests:

• Positive growth control: Inoculate a plate without any antimicrobial strips to confirm that the organism grows under test conditions.
• Negative control: Uninoculated agar plate incubated alongside tests to verify sterility of the medium.
• Reference strains: Use ATCC or other certified reference strains with known MIC ranges for the antimicrobial agents being tested. Common reference strains include:
  • Staphylococcus aureus ATCC 29213
  • Escherichia coli ATCC 25922
  • Pseudomonas aeruginosa ATCC 27853
  • Enterococcus faecalis ATCC 29212
  • Candida parapsilosis ATCC 22019 (for antifungal testing)

The MIC for the reference strain must fall within the established QC range for the test to be valid. If the QC result is out of range, investigate potential causes such as expired strips, contaminated medium, or incorrect inoculum.

Conceptual Workflow

Step 1: Preparation

1. Remove Etest strips from freezer storage and allow to equilibrate to room temperature for 30 minutes.
2. Label agar plates with organism identification, antimicrobial agent(s), date, and technician initials.
3. Prepare bacterial or yeast suspension to 0.5 McFarland standard in sterile saline or broth.
4. Within 15 minutes of preparation, inoculate the agar plate by streaking with a sterile cotton swab in three directions.

Step 2: Strip Application

1. Allow inoculated plate to dry for 10–15 minutes at room temperature with the lid slightly ajar.
2. Using sterile forceps, remove the Etest strip from its foil pouch.
3. Apply the strip to the agar surface with the scale facing upward and the high-concentration end toward the plate edge.
4. Press gently with forceps or applicator to ensure complete contact, avoiding air bubbles.
5. For multiple strips, place them radially at least 15–20 mm apart.

Step 3: Incubation

1. Incubate plates inverted (agar side up) at 35 ± 2°C for 16–24 hours under aerobic conditions.
2. For fastidious organisms, extend incubation as needed (e.g., 24–48 hours for Haemophilus spp., 48 hours for anaerobes).
3. Maintain humidity to prevent agar drying during extended incubation.

Step 4: Reading the Ellipse

1. Examine the plate against a dark background with adequate lighting.
2. Read the MIC at the point where the elliptical inhibition zone intersects the strip scale.
3. For most organisms, read at the complete inhibition edge (no visible growth, microcolonies, or haziness).
4. For organisms with trailing endpoints (e.g., some Candida spp. with azoles), read at the point of 80% inhibition (significant reduction in growth).

Step 5: Interpretation

1. Record the MIC value in µg/mL.
2. Compare the MIC to established clinical breakpoints (CLSI or EUCAST) to determine susceptibility category (S, I, or R).
3. Document results in the laboratory information system or research records.

Quality Checks

Several quality checks ensure reliable Etest results:

• Inoculum verification: Confirm that the 0.5 McFarland suspension produces semi-confluent growth (not too heavy or too light) after incubation.
• Strip integrity: Inspect strips for damage, discoloration, or expiration before use.
• Agar quality: Verify that the agar surface is free from cracks, bubbles, or contamination.
• Incubation conditions: Monitor incubator temperature and CO₂ levels (if applicable) daily.
• QC strain performance: Reference strain MICs must fall within established ranges for the test to be valid.
• Reproducibility: For research applications, perform duplicate tests on separate days to assess reproducibility.

Result Interpretation

Reading the MIC

The MIC is read at the intersection of the elliptical inhibition zone with the strip scale. The ellipse should be symmetrical around the strip. Key reading rules include:

• Complete inhibition: Read at the point where growth stops completely. This applies to most bactericidal agents and many bacteriostatic agents.
• Trailing endpoints: For some organism-antimicrobial combinations (e.g., azoles against Candida spp.), a trailing effect may occur where microcolonies or faint growth appear within the inhibition zone. In such cases, read at the point of 80% inhibition (significant reduction in growth compared to the control area).
• Skip wells or gaps: If there is a clear zone of inhibition followed by growth and then another inhibition zone, read the MIC at the first intersection point.
• Microcolonies: If microcolonies appear within the inhibition zone, read at the point where the majority of microcolonies stop, unless the microcolonies represent resistant subpopulations.

Interpreting the MIC Value

The MIC value is read directly from the strip scale. For example, if the ellipse intersects at the 4 µg/mL mark, the MIC is 4 µg/mL. If the intersection falls between two markings, round up to the next higher value (e.g., between 2 and 4 µg/mL, read as 4 µg/mL).

Compare the MIC to clinical breakpoints established by CLSI or EUCAST. Breakpoints vary by organism, antimicrobial agent, and infection site. For example, for S. pneumoniae and penicillin, the EUCAST non-meningitis breakpoint for susceptibility is ≤0.06 µg/mL, while for meningitis it is ≤0.06 µg/mL with different interpretive criteria [5].

Categorical Agreement

Categorical agreement (CA) refers to the concordance between the Etest result and the reference method (usually BMD) in classifying isolates as susceptible, intermediate, or resistant. Studies have reported CA values ranging from 72% to 98% depending on the organism-antimicrobial combination [1, 2]. Essential agreement (EA) refers to the MIC value falling within ±1 doubling dilution of the reference method. EA values for Etest are typically ≥90% for most combinations, though exceptions exist [5].

Troubleshooting

Limitations

The Etest method has several limitations that users should recognize:

• Cost: Etest strips are more expensive per test than disk diffusion, making them less suitable for high-volume screening.
• Subjective reading: The endpoint determination can be subjective, particularly for trailing endpoints or microcolonies, leading to inter-reader variability.
• Medium dependence: Performance varies with agar brand and lot, as demonstrated by studies showing lower essential agreement on certain media [5].
• Limited antimicrobial range: Not all antimicrobial agents are available as Etest strips, and some combinations may not be validated.
• No automated reading: Unlike automated systems (e.g., Vitek 2, BD Phoenix), Etest requires manual reading and interpretation, which can be time-consuming for large batches.
• Combination testing complexity: While methods exist for testing antimicrobial combinations (e.g., side-by-side or strip cross methods), these require careful interpretation and may not be standardized [1].
• Breakpoint limitations: For uncommon organisms or new antimicrobial agents, clinical breakpoints may not be available, making interpretation challenging [4].

Documentation

Proper documentation is essential for traceability and quality assurance. Record the following information for each Etest:

• Organism identification: Species and source (e.g., clinical isolate, reference strain)
• Antimicrobial agent: Name and concentration range on the strip
• Strip lot number and expiration date
• Agar medium: Type, manufacturer, lot number, and preparation date
• Inoculum: McFarland standard value and preparation method
• Incubation conditions: Temperature, atmosphere, and duration
• MIC value: Read in µg/mL, with any notes on trailing or microcolonies
• Interpretation: Susceptibility category based on applicable breakpoints
• QC results: Reference strain MIC and whether it fell within the acceptable range
• Technician initials and date

For research applications, also document the purpose of the test, experimental design, and any deviations from the standard protocol.

Biosafety Considerations

Etest procedures involve handling live microorganisms and should be performed in accordance with standard microbiological practices for Biosafety Level 1 (BSL-1) or BSL-2 as appropriate [6]. Key biosafety measures include:

• Personal protective equipment: Wear laboratory coats, gloves, and eye protection when handling cultures.
• Work surface: Perform all manipulations on a disinfected bench or within a biological safety cabinet if working with organisms that produce aerosols.
• Waste disposal: Decontaminate all contaminated materials (plates, swabs, strips) by autoclaving before disposal.
• Hand hygiene: Wash hands thoroughly after handling cultures and before leaving the laboratory.
• Spill management: Cover spills with absorbent material, apply disinfectant (e.g., 10% bleach or 70% ethanol), allow contact time, and clean up following institutional protocols.
• Training: Ensure all personnel are trained in safe microbiological practices and understand the risks associated with the organisms being tested [6, 7].

For routine teaching laboratory exercises using BSL-1 organisms (e.g., E. coli ATCC 25922, S. aureus ATCC 29213), standard aseptic technique and basic biosafety precautions are sufficient. Do not use this protocol for clinical isolates of select agents, highly pathogenic organisms, or strains with known virulence factors without appropriate containment and institutional approval.

Frequently Asked Questions

1. How do I read an Etest when the ellipse is not perfectly symmetrical?

An asymmetrical ellipse usually indicates uneven contact between the strip and the agar surface. If the asymmetry is minor, read the MIC at the point where the majority of the inhibition zone intersects the strip scale. If the asymmetry is severe (e.g., one side of the ellipse extends much farther than the other), the result should be considered invalid, and the test should be repeated with careful strip application. Ensure the agar surface is level and the strip is pressed evenly along its entire length.

2. What should I do if microcolonies appear within the inhibition zone?

Microcolonies within the inhibition zone can indicate heteroresistance (a subpopulation of resistant cells) or a mixed culture. First, subculture the isolate to confirm purity. If the culture is pure, read the MIC at the point where the majority of microcolonies stop, but note the presence of microcolonies in the documentation. For clinical decision-making, the presence of microcolonies may indicate emerging resistance, and the result should be interpreted cautiously. Consider repeating the test with a higher inoculum or using a different method (e.g., BMD) for confirmation.

3. Can I use Etest strips on non-standard agar media?

Etest strips are validated for use on specific media, typically Mueller-Hinton agar for bacteria and RPMI 1640 agar for yeasts. Using non-standard media may affect antimicrobial diffusion and organism growth, leading to inaccurate MIC results. If you must use an alternative medium, perform a validation study comparing Etest results on the alternative medium to reference BMD results for a panel of strains with known MICs. Without such validation, results on non-standard media should be considered preliminary.

4. How do I interpret Etest results for antimicrobial combinations?

For combination testing (e.g., aztreonam with ceftazidime-avibactam), two methods have been described: the side-by-side (SS) method, where two strips are placed parallel and 10 mm apart, and the strip cross (SX) method, where one strip is placed perpendicularly on top of the other at a specific concentration mark [1]. The SX method has shown higher categorical agreement with BMD (98%) compared to the SS method (72%) in one study [1]. Interpretation involves assessing whether the combination produces a synergistic effect (e.g., a deformed or truncated inhibition zone). These methods are not yet standardized, and results should be interpreted with caution, ideally in consultation with an infectious disease specialist.

References and Further Reading

• Evaluation of two gradient diffusion tests to determine susceptibility to aztreonam and ceftazidime-avibactam in combination
• Performance of EUCAST disc diffusion and supplementary methods to detect reduced susceptibility to linezolid in enterococci
• Rapid Raman spectroscopy-based test for antimicrobial resistance
• Global Epidemiology of Invasive Infections by Uncommon Candida Species: A Systematic Review
• Multicenter comparison of Etest, Vitek2 and BD Phoenix to broth microdilution for beta-lactam susceptibility testing of Streptococcus pneumoniae
• Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition
• NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules
• NCBI Bookshelf: Molecular Biology and Laboratory Methods

Related Articles

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