E-Test used for detection of susceptibility type and MIC of antibiotic against a pathogenic bacteria

1. Epsilometer test
Dr. Samira Fattah
PhD in Medical Bacteriology
College of Health Sciences-HMU

2. Epsilometer Test
• Abbreviated as E-Test
• Is a Quantitative method of antibiotic susceptibility testing.
• Applies both dilution of antibiotics and diffusion of antibiotic in to the
medium.
• It is a very simple test to perform MIC of the bacterial isolate as
compared to the other techniques like broth and agar dilution methods.

3. Epsilometer Test
• The E-test is a plastic strip (5 x 50 mm; antibiotic carrier)
with a continuous gradient of antibiotic immobilized on
one side and MIC interpretative scale on the other side.
• When transferred to the agar, the continuous antibiotic
gradient established under the strip remains stable over a
period covering the critical times of most microorganisms
subjected to susceptibility testing.

4. Epsilometer Test
E-Test can be classified to 4 categories:
• Antibiotics
• Antifungal
• Anti-mycobacterial
• Resistance phenotype testing

5. Purpose of E-Test
1. Determine the MIC of fastidious, slow-growing or nutritionally
deficient micro-organisms ( like N. gonorrhoeae, Streptococcus
pneumoniae and Haemophilius species), or for a specific type of
patient or infection .
2. Detect low levels of resistance .
3. Confirm/ detect a specific antimicrobial resistance phenotype
such as ESBL, MBL, and AmpC .
4. Test an antimicrobial not performed in routine use or a new,
recently introduced antimicrobial agent.

6. Test requirements
1. Commercially available E-test strips
2. 0.5 McFarland standards,
3. sterile Mueller Hinton agar plates. In a 90 mm plate, a single
antibiotic strip can be tested. In a 150 mm plate, at least 4 antibiotic
strips can be tested.
4. Forceps
5. sterile swabs
6. bacterial suspension adjusted to 0.5 McFarland standards.

7. Method
1. The surface of agar plate inoculated with an adjusted bacterial
suspension by swab in the same manner as a disk diffusion test.
2. Apply strips to agar surface using forceps (or E-test applicator if
available).
3. Place the strip with the scale visible (i.e. facing upwards). Do
not remove or replace a strip once it has touched the agar.
4. If strips stick together, they may be pulled apart by handling the
section marked E. Do not touch any other area of the strip.
5. Incubate Plates at 37°C for 18-24 hrs.
6. After incubation, the interaction of the antimicrobial agent
gradient and the test bacterial inoculum gives rise to elliptical
inhibitory zones.
7. The results are read in the intersection of the ellipse with a MIC
scale (in μg/ml on the strip).

8. Results and interpretation
1. Read plates after the recommended incubation period only if
sufficient growth is seen and the inhibition eclipse is clearly visible.
2. Always read the end point at complete inhibition of all growth
including hazes and isolated colonies.
3. Since E- test comprises a continuous gradient, MIC values in
between two – fold dilutions can be obtained.
4. Always round up these values to the next two-fold dilution before
interpretation. For example: If ampicillin breakpoints are given as
S=1, I = 2, R=4 μg/ml, then an E-test MIC of 1.5 μg/ml is rounded
up to 2 μg/ml and the category reported as Intermediate (I).

9. Problems with E-test reading
Note: printed list is distributed to students