E-TEST (Epsilometer): Principle, Procedure, Results

Epsilometer test (E- test ) is an ‘exponential gradient’ method of determination of antimicrobial resistance. The E-test has been developed to provide a direct quantification of antimicrobial susceptibility of microorganisms . This is a quantitative method that applies both the dilution of antibiotics and diffusion of antibiotics into the medium.

E-Test showing MIC
E-Test showing MIC

The device consists of a predefined, continuous, and exponential gradient of antibiotic concentrations immobilized along a rectangular plastic test strip. After 48 hours of incubation, a drop-shaped inhibition zone intersects the graded test strip at the inhibitory concentration (IC) of the antibiotic.


  • A predefined stable antimicrobial gradient is present on a thin inert non-porous plastic carrier strip 5mm wide, 60 mm long known as E-test strip.
  • When this E test strip is applied onto an inoculated agar plate, there is an immediate release of the drug and the establishment of an antimicrobial concentration gradient in an agar medium.
  • After overnight incubation, the tests are read by viewing the strips from the top of the plate, a symmetrical inhibition ellipse is produced.
  • The intersection of the lower part of the ellipse-shaped growth inhibition area with the test strip indicates the MIC value.


MEDIA: Mueller Hinton agar (MHA) plates (uniform depth of 4 mm)


  • E-test strips (of desired antibiotic)
  • McFarland standard 0.5
  • Forceps
  • Sterile cotton swabs
  • Sterile normal saline, 4 ml volumes in tubes

Bacterial strains
• Non-fastidious cultures plated out for single colonies.
• Strain for quality control: Escherichia coli ATCC 25922


  1. Inoculum Preparation
    1. Remove the E-test package from the freezer (-20°C) at least 30 minutes before required.
    2. Emulsify 3 or 4 individual test strain colonies and transfer to a tube of saline.
    3. Compare turbidity to that in the 0.5 McFarland standards. Adjust turbidity of inoculum to match that standard.
  2. Inoculation in Muller Hinton Agar
    1. Dip a sterile cotton swab into the inoculums and pulling out slightly, rotate the swab several times against the inside of the tube above the fluid level to remove excess liquid.
    2. Streak the swab over the entire surface of the agar plate by rotating the plate approximately 60o. Complete inoculation by running the swab around the rim of the agar.
    3. Leave the lid of the plate ajar for 5 minutes (no more than 15 minutes) to allow any excess moisture to be absorbed before applying strips.
      NOTE: Swab plate within 15 minutes of preparing the adjusted inoculums
  1. Application of E-test strips:
  1. Open E-test package by cutting package along the broken line. Apply strips to agar surface using forceps (or E-test applicator if available).
  2. Place the strip with the ‘E end’ at the edge of the plate and with the scale visible (i.e. facing upwards).
  3. If strips stick together, they may be pulled apart by handling the section marked E. Do not touch any other area of the strip.
  4. Use templates to position 4 to 6 strips onto a 150 mm plate or one (seldom two) strips onto a 90 mm plate. Do not remove or replace a strip once it has touched the agar.
  5. Repeat the entire procedure also for Quality control Strain (E. coli ATCC 25922)
  6. Incubate Plates at 37°C for 18-24 hrs.

Result and Interpretation

A Staphylococcus aureus isolate tested by the Etest gradient diffusion method
A Staphylococcus aureus isolate tested by the E-test gradient diffusion method
  1. Read MIC at the point where the ellipse intersects the scale. If a MIC value between two twofold dilutions is seen, always round up to the highest value.
  2. Read the MIC value at complete inhibition of all growth.
  3. If the intersect differs on either side of the strip, read the MIC as the greater value. Ignore any growth at the edge of the strip.

MIC values of the bacteria should be interpreted as S (Susceptible), I (Intermediate), or R (Resistant) by comparing the breakpoint values of each antibiotic with the criteria recommended by CLSI.


  1. Nachnani, S., Scuteri, A., Newman, M. G., Avanessian, A. B., & Lomeli, S. L. (1992). E-test: a new technique for antimicrobial susceptibility testing for periodontal microorganisms. Journal of periodontology, 63(7), 576–583. https://doi.org/10.1902/jop.1992.63.7.576 
  2. Brown, D. F., & Brown, L. (1991). Evaluation of the E test, a novel method of quantifying antimicrobial activity. The Journal of antimicrobial chemotherapy, 27(2), 185–190. https://doi.org/10.1093/jac/27.2.185

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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