Antimicrobial susceptibility test

Antimicrobial susceptibility test– Kirby Beaur method (according to CLSI guidelines)


In 1961, Ericsson and Sherris in collaboration with the World Health Organization undertook an initiative to define and standardize susceptibility testing on a global level. Experts from 16 countries stretching from the United States across to Japan contributed to this, which, in its time, was a monumental effort to obtain global consensus on the standardization of susceptibility testing with MIC and disk diffusion methods. The findings and recommendations of this project were published in the Acta Pathologica et Microbiologica Scandinavica and titled “Antibiotic sensitivity testing, report from an International Collaborative Study (ICS)”. This comprehensive reference, which has been cited over 5,000 times in the peer reviewed literature, was taken one step further by the Clinical Laboratory Standards Institute (CLSI) formerly NCCLS in the United States to develop specific clinical laboratory standards for antimicrobial susceptibility testing. In the meantime, the Food and Drug Administration (FDA) began its efforts to define the quality specifications of antibiotic disk reagents based on the efforts and findings of Ericsson et al. and to regulate their use for clinical testing.


  1. Bacterial culture
  2. Muller Hinton media plates
  3. Sterile swabs
  4. Markers and scale
  5. Bunsen burner
  6. Antibiotic discs
  7. Test organism


Preparation of Müeller-Hinton Agar

  1. Müeller-Hinton agar should be prepared from a commercially available dehydrated base according to the manufacturer’s instructions.
  2. Immediately after autoclaving, allow it to cool in a 45 to 50oC water bath.
  3. Pour the freshly prepared autoclaved and cooled medium into glass or plastic, flat-bottomed petri dishes on a level, horizontal surface to give a uniform depth of approximately 4 mm. This corresponds to 60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30 ml for plates with a diameter of 100 mm.
  4. The agar medium should be allowed to cool to room temperature and, unless the plate is used the same day, stored in a refrigerator (2 to 8oC).
  5. Plates should be used within seven days after preparation unless adequate precautions, such as wrapping in plastic, have been taken to minimize drying of the agar.
  6. A representative sample of each batch of plates should be examined for sterility by incubating at 30 to 35oC for 24 hours or longer.

Preparation of inoculums

  1. At least three to five well-isolated colonies of the same morphological type are selected from an agar plate culture. The top of each colony is touched with a loop, and the growth is transferred into a tube containing 4 to 5 ml of a suitable broth medium.
  2. The broth culture is incubated at 35oC and left overnight for growth.

Inoculation of Test Plates

  1. Pick 0.1 ml of the culture and spread it on a NA plate.
  2. Let it absorb onto the surface
  3. Now carefully place the a/b discs equidistantly on the plate.
  4. Invert and incubate the plates overnight at the appropriate temperature.

NOTE: Extremes in inoculum density must be avoided. Never use undiluted overnight broth cultures or other unstandardized inocula for streaking plates.

Reading Plates and Interpreting Results

  1. After 16 to 18 hours of incubation, each plate is examined. If the plate was satisfactorily streaked, and the inoculum was correct, the resulting zones of inhibition will be uniformly circular and there will be a confluent lawn of growth. If individual colonies are apparent, the inoculum was too light and the test must be repeated. The diameters of the zones of complete inhibition (as judged by the unaided eye) are measured, including the diameter of the disc. Zones are measured to the nearest whole millimeter, using sliding calipers or a ruler, which is held on the back of the inverted petri plate. The petri plate is held a few inches above a black, nonreflecting background and illuminated with reflected light. If blood was added to the agar base (as with streptococci), the zones are measured from the upper surface of the agar illuminated with reflected light, with the cover removed. If the test organism is a Staphylococcus or Enterococcus spp., 24 hours of incubation are required for vancomycin and oxacillin, but other agents can be read at 16 to 18 hours. Transmitted light (plate held up to light) is used to examine the oxacillin and vancomycin zones for light growth of methicillin- or vancomycin- resistant colonies, respectively, within apparent zones of inhibition. Any discernable growth within zone of inhibition is indicative of methicillin or vancomycin resistance.
  2. The zone margin should be taken as the area showing no obvious, visible growth that can be detected with the unaided eye. Faint growth of tiny colonies, which can be detected only with a magnifying lens at the edge of the zone of inhibited growth, is ignored. However, discrete colonies growing within a clear zone of inhibition should be subcultured, re-identified, and retested. Strains of Proteus spp. may swarm into areas of inhibited growth around certain antimicrobial agents. With Proteus spp., the thin veil of swarming growth in an otherwise obvious zone of inhibition should be ignored. When using blood-supplemented medium for testing streptococci, the zone of growth inhibition should be measured, not the zone of inhibition of hemolysis. With trimethoprim and the sulfonamides, antagonists in the medium may allow some slight growth; therefore, disregard slight growth (20% or less of the lawn of growth), and measure the more obvious margin to determine the zone diameter.
  3. The sizes of the zones of inhibition are interpreted by referring to Tables in CLSI interpretative criteria table.
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