Epsilometer Test (E test)

Assignment no 2
Clinical Microbiology

Submitted by: Aryan Imran

Submitted to: Ma'am Saba
Roll no: 075
Semester: 7th
Session: “B”
Department: Medical Laboratory Technology
Topic: Epsilometer Test (E test)

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 Epsilometer Test (E test)

Epsilometer Test (E Test)

Introduction

E test is a quantitative technique for determining the antimicrobial susceptibility of

Gram-negative and Gram-positive aerobic bacteria such as Enterobacteriaceae, Pseudomonas,
Staphylococcus and Enterococcus species and fastidious bacteria, such as anaerobes, N.
gonorrhoeae, S. pneumoniae, Streptococcus and Haemophilus species. The system comprises a
predefi ned antibiotic gradient which is used to determine the Minimum Inhibitory Concentration
(MIC), in μg/mL, of different antimicrobial agents against microorganisms as tested on agar
media using overnight incubation.

Purpose of E test

1. Determine the MIC of fastidious, slow-growing or nutritionally deficient micro-

organisms, or for a specific type of patient or infection.
2. Detect low levels of resistance.
3. Confirm an equivocal Antimicrobial Susceptibility Test result.
4. Test an antimicrobial not performed in routine use or a new, recently introduced
antimicrobial agent.

Principle of E test

E test is a quantitative technique that is based on combination of concept of both dilution

and diffusion principle for susceptibility testing. E test strip is placed on to an inoculated agar
plate; there is an immediate release of antibiotics from the plastic carrier surface into the agar
surface. After incubation, bacterial growth becomes visible, symmetrical inhibition ellipse along
the strip is seen. The MIC value is read from the scale in terms of µg/ml where the ellipse edge
intersects the strip.

Procedure

1. Remove the E-test package from the freezer (-20°C) and kept at room temperature at least 30
minutes before the test performed.
2. Emulsify several well-isolated test strain colonies from an overnight agar plate in saline.
3. Vortex for 15 second.
4. Adjust the suspension turbidity to 0.5 McFarland standards. For mucoid organisms, adjust
suspension to 1 McFarland standard.
5. Soak a sterile cotton swab into the inoculum suspension and remove the excess fluid by
pressing it against the inside wall of the test tube.
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 Epsilometer Test (E test)

6. Streak the swab over the entire agar surface in 3 directions by rotating the plate 60 ⁰ on
Mueller Hinton Agar (MHA).
7. Allow the plate to dry for 5-15 minutes so that the surface is completely dry before applying
E test gradient strip.
8. Place the E test strip on agar plate with MIC scale facing upward and the concentration
maximum nearest the rim of the plate.
9. Incubate the plate. After incubation observe zone of inhibition.

Results and Interpretation

 Read the MIC value at the point where ellipse intersects the scale/E-test strip.
 If the MIC value between the standard two-fold dilution is seen, always round up to the
highest value.
 Read the MIC value at complete inhibition of all growth including isolated colonies.
 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.
 Interpret E-test MIC results as Susceptible, Intermediate or Resistant.

Precautions

 Aseptic procedures and precautions against microbiological hazards should be used when
handling bacterial specimens.
 E-test should be used strictly according to the procedures described.

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 Epsilometer Test (E test)

 One should be very careful while placing the E strip on the plate containing the bacterial
suspension to not touching the edge of the plate.
 Read results only if a good inhibition ellipse is visible.
 While doing or placing multiple strips on a single plate, the strips shouldn’t touch one
another that would make the difference in the result interpretation of the given antibiotic.

Advantages of E test

 Easy to perform and requires minimal training for test performance, however, end points
can be difficult to determine for the azoles against Candida
 Contamination can be easily recognized.
 Can be easily set up for a small number of clinical isolates.
 Adequate method to detect potentially resistant strains to Amphotericin B.

Limitations of E test

 E-test is not suitable for Cryptococcus neoformans.