Dilution Tests for Antibiotic Sensitivity Testing (Broth dilution, Agar dilution and E-Test)

  • Performed to determine the minimum inhibitory concentration (MIC) of an antimicrobial agent.
  • MIC is defined as the lowest concentration of an antimicrobial agent that inhibits the growth of organisms.

Estimation of the MIC is useful to:

Regulate the therapeutic dose of the antibiotic accurately in the treatment of many life-threatening situations, such as bacterial endocarditis.

Test antimicrobial sensitivity patterns of slow-growing bacteria, such as M. tuberculosis.

Following methods are carried out to determine the MIC

  • Broth dilution method
  • Agar dilution method
  • Epsilometer test (E-test)

Broth Dilution Method

Quantitative method for determining the MIC of an antimicrobial agent that inhibits the growth of organisms in vitro.

In this method, the antimicrobial agent is serially diluted in Mueller–Hinton broth by doubling dilution in tubes and then a standard suspension of the broth culture of test organism is added to each of the antibiotic dilutions and control tube.

This is mixed gently and incubated at 37°C for 16–18 hours.

An organism of known susceptibility is included as a control.

The MIC is recorded by noting the lowest concentration of the drug at which there is no visible growth as demonstrated by the lack of turbidity in the tube.

The main advantage of this method is that this is a simple procedure for testing a small number of isolates. The added advantage is that using the same tube, the minimum bactericidal concentration (MBC) of the bacteria can be determined.

  • The MBC is determined by subculturing from each tube, showing no growth on a nutrient agar without any antibiotics.
  • Subcultures are made from each tube showing no growth into the nutrient agar plates without any antibiotics.
  • The plates are examined for growth, if any, after incubation overnight at 37°C.
  • The tube containing the lowest concentration of the drug that fails to show any growth on subculture plate is considered as the MBC of the antibiotic for that strain.

Broth microdilution is done using microtiter plates and is considered the “gold standard.”

Agar dilution method

  • Quantitative method for determining the MIC of antimicrobial agent against the test organism.
  • Mueller–Hinton agar is used in this method.
  • Serial dilution of the antibiotic are made in agar and poured onto Petri dishes.
  • Dilutions are made in distilled water and added to the agar that has been melted and cooled to not more than 60°C.
  • One control plate is inoculated without antibiotics.
  • Organism to be tested is inoculated and incubated overnight at 37°C.
  • Plates are examined for presence or absence of growth of the bacteria.
  • The concentration at which bacterial growth is completely inhibited is considered as the MIC of the antibiotic.
  • The organisms are reported sensitive, intermediate, or resistant by comparing the test MIC values with that given in CLSI guidelines.

The main advantage of the method is that a number of organisms can be tested simultaneously on each plate containing an antibiotic solution.

Epsilometer test (E test)

  • Based on the principle of disc diffusion, is an automated system for measuring MIC of a bacterial isolate. In this method, an absorbent plastic strip with a continuous gradient of antibiotic is immobilized on one side.
  • MIC interpretative scale corresponding to 15 twofold MIC dilutions is used on the other side.
  • The strip is placed on the agar plate inoculated with the test organism with the MIC scale facing toward the opening side of the plate.
  • An elliptical zone of growth inhibition is seen around the strip after incubation at 37°C overnight.
  • The MIC is read from the scale at the intersection of the zone with the strip.
  • The end point is always read at complete inhibition of all growth including hazes and isolated colonies.

E test is a very useful test for easy interpretation of the MIC of an antibiotic.

Binod G C

I'm Binod G C (MSc), a PhD candidate in cell and molecular biology who works as a biology educator and enjoys scientific blogging. My proclivity for blogging is intended to make notes and study materials more accessible to students.

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