E-Test in Microbiology: A Quantitative Method for MIC Determination

The E-test is a quantitative antimicrobial susceptibility testing method that uses a plastic strip impregnated with a predefined concentration gradient of an antimicrobial agent to determine the minimum inhibitory concentration (MIC) directly on an agar plate. The method combines the simplicity of disk diffusion setup with the quantitative output of broth or agar dilution reference methods. This article explains the principle, procedure, interpretation, quality control, and limitations of the E-test for laboratory students, technicians, researchers, and diagnostic professionals who need to generate reliable MIC data for clinical or research decisions.

At a Glance

The E-test is a gradient diffusion method that produces a continuous MIC value instead of a categorical susceptible, intermediate, or resistant result. It is set up like a disk diffusion test but uses a calibrated plastic strip instead of a paper disk. After incubation, an elliptical zone of inhibition forms around the strip, and the MIC is read where the growth edge intersects the graded scale on the strip.

The E-test was evaluated against conventional agar dilution MIC methods in a study of ten antimicrobial agents and a variety of bacterial organisms. Correlation between MICs by the agar dilution and E-test methods was good, with 98.85% of results within 2 log2 dilution steps in a total of 1304 tests. The E-test is technically straightforward because tests are set up in the same way as the disc diffusion method. The versatility and ease of use of the E-test make the method an attractive alternative to conventional dilution tests [6].

Principle of the E-Test

The E-test strip is a thin plastic carrier with a predefined continuous gradient of an antimicrobial agent immobilized on one surface. The gradient is calibrated to cover a range of concentrations that spans clinically relevant MIC values. When the strip is placed on an inoculated agar plate, the antimicrobial agent diffuses into the agar immediately, establishing a stable concentration gradient in the surrounding medium.

Bacterial growth occurs across the plate except where the local antimicrobial concentration exceeds the MIC of the organism. After incubation, an elliptical zone of inhibition develops around the strip. The point where the growth margin intersects the strip corresponds to the MIC, which is read directly from the printed scale on the strip.

The E-test is based on diffusion of a pre-formed antibiotic gradient from a plastic strip [6]. This design allows the method to produce a continuous MIC value instead of a discrete value from a two-fold dilution series. The continuous scale can reveal MIC values that fall between standard dilution steps, which can be clinically useful for detecting subtle changes in susceptibility.

Comparison with Other Susceptibility Testing Methods

Agar Dilution and Broth Microdilution

Agar dilution and broth microdilution are reference methods that use two-fold serial dilutions of an antimicrobial agent in agar or broth. These methods produce discrete MIC values that correspond to the lowest concentration that inhibits visible growth. They are labor intensive and require preparation of dilution series, which makes them practical for batch testing but inefficient for testing individual clinical isolates.

The E-test produces a continuous MIC value and requires no dilution series preparation. In a comparative evaluation, correlation between MICs by the agar dilution and E-test methods was good, with 98.85% of results within 2 log2 dilution steps in a total of 1304 tests [6]. The E-test is less labor intensive than broth dilution methods and may be useful for testing individual clinical isolates [13].

Disk Diffusion

Disk diffusion is a qualitative method that produces a zone of inhibition diameter, which is interpreted as susceptible, intermediate, or resistant using breakpoint tables. The method is simple and inexpensive but does not produce a quantitative MIC value.

The E-test is set up in the same way as the disc diffusion method [6]. This similarity makes the E-test easy to adopt in laboratories already performing disk diffusion. The key difference is that the E-test strip contains a continuous gradient instead of a single concentration, allowing quantitative MIC determination.

Automated Systems

Automated susceptibility testing systems can identify organisms and determine susceptibility in a single workflow. However, studies have shown that automated systems can produce errors when compared with reference methods. In one study of piperacillin-tazobactam susceptibility testing in ceftriaxone non-susceptible Enterobacterales, the VITEK 2 system had a very major error rate of 26.7% when compared with broth microdilution, while the Etest had no very major errors but a major error rate of 6.3% [20].

Some automated systems used in clinical microbiology laboratories are able to detect products responsible for antimicrobial resistance. In a study of 626 isolates examined for presumptive detection of extended-spectrum beta-lactamase production, the E-test detected ESBL production in isolates that were not warned as being ESBL producers by the automated system. The data suggest that clinical microbiology laboratories should also rely on rapid automated systems but also use another method for screening ESBL producers, such as the E-test [7].

Applications of the E-Test

Antibacterial Susceptibility Testing

The E-test is widely used for determining MICs of antibacterial agents against a variety of bacterial pathogens. It is particularly useful for organisms that are slow growing, fastidious, or require specialized media. The method has been evaluated for nutritionally variant streptococci, where E-test with isosensitest agar supplemented with 5% defibrinated horse blood and 0.001% pyridoxal HCl gave complete agreement within one twofold-dilution titer of the agar reference method [12].

Antifungal Susceptibility Testing

The E-test has been evaluated for antifungal susceptibility testing of yeasts. In a study comparing fluconazole and itraconazole MICs obtained by E-test with a microbroth dilution method performed to NCCLS guidelines, E-test results exhibited good correlation with the broth microdilution, with 82% and 81% agreement within +/- 1 two-fold dilution, respectively. The E-test is easy to perform and generates MICs for Candida species in 24 hours [13].

The E-test is also one of the three most frequently used methodologies for evaluating antifungal combinations, along with the checkerboard method and time-kill curves [9].

Mycobacterial Susceptibility Testing

The E-test has been evaluated as an alternative method for susceptibility testing of Mycobacterium tuberculosis. In a study of 100 isolates tested for isoniazid, rifampin, streptomycin, and ethambutol susceptibility using an indirect-proportion method and the E-test method, categorical agreement between the methods was 100% for all four agents. The E-test method appears to be an alternative method to agar proportion for testing the susceptibility of M. tuberculosis isolates to first-line antituberculous agents [8].

ESBL Detection

The E-test can be used for presumptive detection of extended-spectrum beta-lactamase production using strips containing ceftazidime and ceftazidime-clavulanate. In a study of 626 isolates, ESBL production was detected in 26 E. coli strains, 60 K. pneumoniae strains, and 15 K. oxytoca strains by ceftazidime/ceftazidime-clavulanate E-test [7].

Synergy Testing

The E-test has been evaluated for detecting antibiotic synergy against Pseudomonas aeruginosa. In a study comparing the E-test with the checkerboard technique for detecting synergy or antagonism of antibiotic combinations, the agreement between checkerboard and E-test results was 71.2%. The E-test was preferable to the checkerboard method for total cost, with a cost of 8.60 euros per test compared with 21.80 euros per test for the checkerboard method [11].

Colistin Susceptibility Testing

The E-test has been compared with broth microdilution for testing colistin susceptibility in Acinetobacter baumannii. In a study of 115 clinical isolates, a categorical agreement of 98.2% was found, with only two very major errors. However, there was poor concordance at extreme dilutions, with higher MICs by the E-test method. Complete agreement was found for strains for which MICs fell within the range of 0.25 to 1 microg of colistin per ml [10].

Practical Workflow for Performing an E-Test

Step 1: Prepare the Inoculum

Prepare a standardized bacterial suspension from an overnight culture. The turbidity should match a 0.5 McFarland standard, which corresponds to approximately 1 to 2 x 10^8 colony-forming units per milliliter for most bacteria. Use sterile saline or broth as the suspension medium. For fastidious organisms, follow the specific media and supplementation requirements for the organism being tested.

Step 2: Inoculate the Agar Plate

Use a sterile swab to spread the bacterial suspension evenly across the entire surface of the agar plate. Rotate the plate approximately 60 degrees between swabbing directions to ensure complete and even coverage. Allow the plate to dry for a few minutes so that excess moisture is absorbed before applying the strips.

Step 3: Apply the E-Test Strips

Use sterile forceps or the applicator provided by the manufacturer to place the E-test strip on the inoculated agar surface. Press gently to ensure complete contact between the strip and the agar. The strip should be placed with the graded scale facing upward and the MIC scale readable from the top. Multiple strips can be placed on a single plate, arranged radially like spokes of a wheel, provided the zones of inhibition do not overlap.

Step 4: Incubate

Incubate the plate under the appropriate conditions for the organism being tested. Most bacteria require 18 to 24 hours at 35 to 37 degrees Celsius. Fastidious organisms may require supplemented media, increased carbon dioxide, or longer incubation. For antifungal testing, incubation conditions follow the recommendations for the specific organism and antifungal agent.

Step 5: Read the MIC

After incubation, examine the plate for the elliptical zone of inhibition around the strip. The MIC is read at the point where the growth margin intersects the graded scale on the strip. Read the value at the complete inhibition edge, ignoring isolated colonies or faint haze within the zone unless the testing protocol specifies otherwise.

Interpretation of E-Test Results

Reading the Intersection Point

The MIC is the value on the strip scale at the point where the edge of the elliptical zone of inhibition crosses the strip. The growth edge should be read at the point of complete inhibition. For most organisms, this is a sharp demarcation. Some organisms produce a haze or trailing growth that can make the endpoint difficult to read.

Handling Indeterminate Endpoints

Certain organisms and antimicrobial combinations produce endpoints that are difficult to interpret. For example, use of Mueller-Hinton and Columbia-based supplemented agar for nutritionally variant streptococci showed hazy growth and double zoning around the endpoint [12]. When the endpoint is unclear, the test should be repeated or confirmed with a reference method.

Categorical Interpretation

Once the MIC is determined, it can be interpreted as susceptible, intermediate, or resistant using clinical breakpoints published by organizations such as CLSI or EUCAST. The MIC value can also be compared with epidemiological cutoff values to distinguish wild-type from non-wild-type populations.

Agreement with Reference Methods

The E-test generally shows good agreement with reference methods. In a study of piperacillin-tazobactam susceptibility testing, the Etest had a major error rate of 6.3% but no very major errors when compared with broth microdilution [20]. In a study of colistin susceptibility testing in Acinetobacter baumannii, a categorical agreement of 98.2% was found, with only two very major errors [10].

Quality Control and Quality Assurance

Reference Strains

Quality control testing should be performed with reference strains that have established MIC ranges for the antimicrobial agents being tested. These strains should be tested on a regular schedule, typically daily or weekly depending on testing volume and regulatory requirements. The results should fall within the established acceptable ranges for the organism and antimicrobial agent combination.

Internal Quality Control Panels

Total laboratory automation systems can incorporate internal quality control panels that monitor all culture-based processes, including MIC determination by broth microdilution and E-test strips. In one study of an integrated internal quality control panel implemented over 6 months, no errors were detected regarding all culture-based and antimicrobial susceptibility testing processes, with the exception of one major error related to a MIC misreading for imipenem [17].

Media Quality

The quality of the agar medium is critical for E-test performance. The medium must support adequate growth of the test organism and must be free of antimicrobial antagonists. Mueller-Hinton agar is the standard medium for most non-fastidious bacteria. Supplemented media may be required for fastidious organisms, as demonstrated by the need for pyridoxal hydrochloride supplementation for nutritionally variant streptococci [12].

Storage and Handling of Strips

E-test strips must be stored according to manufacturer instructions, typically refrigerated and protected from moisture. Strips should be allowed to reach room temperature before opening the container to prevent condensation. Expired strips must not be used.

Records and Documentation

What to Record

For each E-test performed, record the following information:

• Patient or sample identifier
• Organism identification
• Antimicrobial agent tested
• MIC value read from the strip
• Interpretation category based on breakpoints
• Date and time of test setup and reading
• Technician who performed the test
• Quality control results for the batch or day

Documentation for Clinical Decisions

MIC values from E-tests are used for clinical decisions, so documentation must be complete and traceable. The laboratory should maintain records that allow results to be traced back to the specific strip lot, media lot, and quality control results. This traceability supports investigation of unexpected results and compliance with quality management requirements.

Documentation for Research

For research applications, record the exact protocol used, including media formulation, inoculum preparation, incubation conditions, and reading criteria. This documentation supports reproducibility and comparison of results across studies. The lack of a harmonized reference assay for some applications, such as essential oil MIC determination, highlights the importance of detailed protocol documentation [14].

Common Failure Patterns and Troubleshooting

No Zone of Inhibition

If no zone of inhibition forms around the strip, possible causes include failure of the antimicrobial agent to diffuse, contamination of the strip, or an organism that is highly resistant. Check the quality control results for the strip lot and repeat the test with a reference strain to verify strip performance.

Zone Too Large or Too Small

An excessively large zone may indicate that the inoculum was too light. An excessively small zone may indicate that the inoculum was too heavy or that the organism grew poorly. Repeat the test with a properly standardized inoculum.

Hazy Growth or Double Zoning

Some organisms produce hazy growth or double zoning around the endpoint, making the MIC difficult to read. This has been observed with nutritionally variant streptococci on certain media [12]. If the endpoint is unclear, repeat the test on a different medium or confirm with a reference method.

Skewed or Distorted Zones

A skewed or distorted zone may indicate that the strip was not in complete contact with the agar or that the agar surface was too wet when the strip was applied. Ensure the agar surface is dry before applying the strip and press the strip gently to ensure complete contact.

Growth Along the Strip Edge

Growth along the edge of the strip can occur if the strip was not pressed firmly onto the agar. This can create a false reading. Ensure complete contact between the strip and the agar surface.

Limitations of the E-Test

Agreement at Extreme Dilutions

The E-test may show poor concordance with reference methods at extreme dilutions. In a study of colistin susceptibility testing in Acinetobacter baumannii, there was poor concordance at extreme dilutions, with higher MICs by the E-test method [10]. Laboratories should be cautious when interpreting E-test MICs that fall at the extremes of the strip range.

Major Errors with Certain Organism-Drug Combinations

The E-test can produce major errors with certain organism-drug combinations. In a study of piperacillin-tazobactam susceptibility testing, the Etest had a major error rate of 6.3% [20]. Laboratories should be aware of the error rates for the specific organism-drug combinations they test and confirm critical results with a reference method when appropriate.

Not a Reference Method

The E-test is not a reference method. It is an alternative method that shows good correlation with reference methods for many organism-drug combinations [6]. For critical clinical decisions, results should be confirmed with a reference method when there is any doubt about the accuracy of the E-test result.

Cost Considerations

The E-test is more expensive per test than disk diffusion or broth microdilution for batch testing. However, it can be more cost-effective for testing individual clinical isolates because it requires less labor and no dilution series preparation [13]. In a study of synergy testing, the E-test was preferable to the checkerboard method for total cost, including reagent cost and technologist time [11].

Biosafety Considerations

Handling of Clinical Isolates

E-test procedures involve handling of viable microorganisms, including potentially pathogenic bacteria and fungi. All work should be performed in a biological safety cabinet when aerosol-generating procedures are performed. Follow the biosafety guidelines for the risk group of the organism being tested, as described in the WHO Laboratory Biosafety Manual [2].

Waste Disposal

Inoculated plates and used E-test strips should be disposed of as biohazardous waste. Autoclave or incinerate contaminated materials according to laboratory waste management protocols.

Personal Protective Equipment

Wear appropriate personal protective equipment, including laboratory coat, gloves, and eye protection, when performing E-test procedures. Wash hands after handling clinical specimens and cultures.

Quality Management System Integration

Standard Operating Procedures

The E-test procedure should be documented in a standard operating procedure that includes the principle, equipment, reagents, step-by-step instructions, quality control requirements, and interpretation criteria. The standard operating procedure should be reviewed and approved by the laboratory director or quality manager.

Training and Competency Assessment

Personnel performing E-tests should receive training on the procedure and demonstrate competency before performing tests independently. Competency should be assessed periodically, including the ability to read endpoints accurately and interpret results correctly.

Proficiency Testing

Laboratories should participate in external proficiency testing programs that include E-test challenges. Proficiency testing results should be reviewed and corrective action taken when results are unsatisfactory. The WHO Laboratory Quality Management System Handbook provides guidance on establishing and maintaining quality management systems in laboratories [1].

Method Validation

Before implementing the E-test for clinical use, the laboratory should validate the method for the organism-drug combinations it will test. Validation should include comparison with a reference method and assessment of accuracy, precision, and reproducibility. The FDA Bioanalytical Method Validation Guidance provides a framework for validation of analytical methods [4].

Professional Escalation Criteria

When to Confirm with a Reference Method

Confirm E-test results with a reference method in the following situations:

• The MIC falls at the extreme of the strip range
• The endpoint is difficult to read due to hazy growth or double zoning
• The result is critical for a patient management decision and the error rate for the organism-drug combination is known to be significant
• The result does not correlate with the organism identification or clinical presentation

When to Repeat the Test

Repeat the E-test when:

• Quality control results for the day are out of range
• The inoculum was not properly standardized
• The plate was over-incubated or under-incubated
• The strip was not in complete contact with the agar
• The zone of inhibition is distorted or unreadable

When to Escalate to a Supervisor

Escalate to a laboratory supervisor when:

• Repeated quality control failures occur with a specific strip lot
• Unexpected resistance patterns are observed that may indicate a testing error or an emerging resistance mechanism
• The E-test result conflicts with results from other testing methods
• The result has significant clinical implications and there is any doubt about its accuracy

Frequently Asked Questions

What is the difference between the E-test and disk diffusion?

The E-test uses a plastic strip with a continuous gradient of antimicrobial agent and produces a quantitative MIC value. Disk diffusion uses a paper disk with a single concentration of antimicrobial agent and produces a qualitative zone diameter that is interpreted as susceptible, intermediate, or resistant. The E-test is set up in the same way as the disc diffusion method, making it easy to adopt in laboratories already performing disk diffusion [6].

How is the MIC read from an E-test strip?

The MIC is read at the point where the edge of the elliptical zone of inhibition intersects the graded scale on the strip. The value at this intersection is the MIC. Read the value at the complete inhibition edge, ignoring isolated colonies or faint haze within the zone unless the testing protocol specifies otherwise.

Can the E-test be used for antifungal susceptibility testing?

Yes, the E-test has been evaluated for antifungal susceptibility testing of yeasts. In a study comparing fluconazole and itraconazole MICs obtained by E-test with a microbroth dilution method, E-test results exhibited good correlation with the broth microdilution, with 82% and 81% agreement within +/- 1 two-fold dilution, respectively. The E-test is easy to perform and generates MICs for Candida species in 24 hours [13].

Can the E-test be used for Mycobacterium tuberculosis susceptibility testing?

Yes, the E-test has been evaluated as an alternative method for susceptibility testing of Mycobacterium tuberculosis. In a study of 100 isolates tested for isoniazid, rifampin, streptomycin, and ethambutol susceptibility, categorical agreement between the indirect-proportion method and the E-test method was 100% for all four agents [8].

What are the limitations of the E-test for colistin susceptibility testing?

The E-test shows good categorical agreement with broth microdilution for colistin susceptibility testing in Acinetobacter baumannii, with a categorical agreement of 98.2% in one study. However, there was poor concordance at extreme dilutions, with higher MICs by the E-test method. Complete agreement was found for strains for which MICs fell within the range of 0.25 to 1 microg of colistin per ml [10].

How does the E-test compare with automated susceptibility testing systems?

The E-test can detect resistance that automated systems miss. In a study of ESBL detection, the E-test detected ESBL production in isolates that were not warned as being ESBL producers by the automated system. The data suggest that clinical microbiology laboratories should also rely on rapid automated systems but also use another method for screening ESBL producers, such as the E-test [7]. In a study of piperacillin-tazobactam susceptibility testing, the VITEK 2 system had a very major error rate of 26.7% while the Etest had no very major errors [20].

What media are recommended for E-testing fastidious organisms?

Fastidious organisms may require supplemented media. For nutritionally variant streptococci, isosensitest agar supplemented with 5% defibrinated horse blood and 0.001% pyridoxal HCl gave complete agreement within one twofold-dilution titer of the agar reference method. Use of Mueller-Hinton and Columbia-based supplemented agar showed hazy growth and double zoning around the endpoint [12].

How should quality control be performed for the E-test?

Quality control should be performed with reference strains that have established MIC ranges for the antimicrobial agents being tested. Test these strains on a regular schedule and verify that results fall within the established acceptable ranges. Internal quality control panels can monitor all culture-based processes, including MIC determination by E-test strips [17].

Related Diagnostic Guides

• How to Perform a Catalase Test: Principle, Procedure, and Interpretation
• How to Perform an Etest for MIC Determination: Protocol and Interpretation
• How to Perform an Indole Test: Principle, Procedure, and Interpretation
• How to Perform an Oxidase Test: Principle, Procedure, and Interpretation
• Starch Hydrolysis Test: Principle, Protocol, and Interpretation

References and Further Reading

• Laboratory Quality Management System Handbook. World Health Organization.
• Laboratory Biosafety Manual. World Health Organization.
• Assay Guidance Manual. National Center for Advancing Translational Sciences.
• Bioanalytical Method Validation Guidance. U.S. Food and Drug Administration.
• NCBI Literature Resources. National Center for Biotechnology Information.
• Evaluation of the E test, a novel method of quantifying antimicrobial activity.. The Journal of antimicrobial chemotherapy, 1991.
• Comparison of the E-test method with an automated bacterial identification and antimicrobial susceptibility detection system for screening extended-spectrum beta-lactamase producers.. Scandinavian journal of infectious diseases, 2003.
• E-test: an alternative method for susceptibility testing of Mycobacterium tuberculosis.. Medical principles and practice : international journal of the Kuwait University, Health Science Centre, 2005.
• Antifungal combinations.. Methods in molecular medicine, 2005.
• Reliability of the E-test method for detection of colistin resistance in clinical isolates of Acinetobacter baumannii.. Journal of clinical microbiology, 2005.
• E-test method for detecting antibiotic synergy against Pseudomonas aeruginosa from neutropenic patients: a cost-effective approach.. The new microbiologica, 2004.
• Evaluation of E test as a rapid method for determining MICs for nutritionally variant streptococci.. Journal of clinical microbiology, 1994.
• Evaluation of the E test system versus a microtitre broth method for antifungal susceptibility testing of yeasts against fluconazole and itraconazole.. The Journal of antimicrobial chemotherapy, 1995.
• Protocol for determining minimum inhibitory concentrations of essential oils against bacterial pathogens using broth microdilution.. 2026.
• Employment of a Resazurin Viability-Based Assay for Minimum Inhibitory and Bactericidal Concentration Determination.. 2026.
• Determination of Fosfomycin MIC values in multidrug-resistant Klebsiella pneumoniae isolates and a cost-effective method attempt for routine laboratory testing.. 2026.
• Total laboratory automation-based monitoring processes: setup and validation of an integrated internal quality control panel.. 2026.
• Proposal for screening cut-off values and use of tolerance disk test (TDtest) for detection of tolerance/persistence to ceftazidime-avibactam in Klebsiella pneumoniae.. 2026.
• Development and Evaluation of Liposomal Nanobiotics for Combating Antibiotic Resistance Among Enterobacteriaceae.. 2026.
• Establishing piperacillin-tazobactam susceptibility in ceftriaxone non-susceptible Enterobacterales: comparing disk diffusion, Etest, and VITEK 2 automated MIC measurements vs. broth microdilution.. Clinical Microbiology and Infection, 2024.
• Comparison of disk diffusion test and Etest for voriconazole and fluconazole susceptibility testing.. Folia Microbiologica (Prague), 2008.
• Antifungal Activity of Natural Compounds vs. Candida spp.: A Mixture of Cinnamaldehyde and Eugenol Shows Promising In Vitro Results. Antibiotics, 2022.
• High prevalence of multidrug-resistant non-fermentative Gram-negative bacilli harboring blaIMP-1 and blaVIM-1 metallo-beta-lactamase genes in Birjand, south-east Iran. Iranian Journal of Microbiology, 2021.
• Fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in hospital and community settings in Chad. Antimicrobial Resistance and Infection Control, 2019.
• Comparative study of the susceptibility of Streptococcus pneumoniae to penicillin G by the E-test and plate dilution methods. Revista Espanola De Quimioterapia, 1994.
• Comparison of Carbapenem Resistance Detected by the BD Phoenix Automated System in Enterobacteriaceae Isolates with E-Test Method. Electronic Journal of General Medicine, 2022.
• Identification of methicillin-resistant staphylococcus aureus with high minimum inhibitory concentrations for vancomycin by E-test and automated methods. Infectio, 2013.
• Comparative Analysis of the Vitek 2 Antifungal Susceptibility System and E-test with the CLSI M27-A3 Broth Microdilution Method for Susceptibility Testing of Indian Clinical Isolates of Cryptococcus neoformans. Mycopathologia, 2012.

This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.