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Microbiology

Susceptibility Testing

This section covers three methods for antimicrobial susceptibility testing: microbroth dilution, disk diffusion, and gradient diffusion

Microbroth Dilution

  • Method: This involves preparing serial dilutions of an antibiotic in a broth medium within microtiter plates. A standardized bacterial inoculum is added to each well, and the plates are incubated. The MIC (Minimum Inhibitory Concentration) is determined by visually identifying the lowest antibiotic concentration that prevents visible bacterial growth
  • Theory: The method is based on exposing bacteria to a range of antibiotic concentrations, with the antibiotic inhibiting bacterial growth by interfering with essential cellular processes. The MIC represents the quantitative measure of the antibiotic’s effectiveness
  • Interpretation: The MIC value is read directly from the microtiter plate and is expressed as a concentration (e.g., µg/mL). The MIC is compared to established interpretive criteria (e.g., CLSI guidelines) to categorize the bacteria as susceptible (S), intermediate (I), or resistant (R)
  • Application: This is a quantitative method, providing precise MIC values. It guides antibiotic selection and dosage optimization, is used to monitor resistance trends, and is highly standardized. It can be labor-intensive and requires specialized equipment

Disk Diffusion

  • Method: A standardized bacterial suspension is spread on a Mueller-Hinton agar plate. Antibiotic-impregnated disks are placed on the agar surface. After incubation, the zone of inhibition (area of no bacterial growth) around each disk is measured
  • Theory: Antibiotics diffuse from the disks into the agar, inhibiting the growth of susceptible bacteria. The size of the zone of inhibition is inversely proportional to the MIC
  • Interpretation: The zone diameter is measured in millimeters (mm). The measured zone diameter is compared to interpretive criteria (e.g., CLSI) to categorize the bacteria as susceptible (S), intermediate (I), or resistant (R)
  • Application: This is a simpler and cost-effective method. It is used to guide antibiotic therapy and monitor resistance patterns. This method provides qualitative or semi-quantitative results

Gradient Diffusion

  • Method: A standardized bacterial suspension is spread on a Mueller-Hinton agar plate. A test strip containing a predefined, continuous gradient of an antibiotic is placed on the agar. After incubation, the MIC value is read directly from the strip at the point where the zone of inhibition intersects the strip
  • Theory: The test strip creates a concentration gradient of the antibiotic in the agar. The point where the bacterial growth is inhibited corresponds to the MIC value
  • Interpretation: The MIC value is read directly from the test strip and is expressed as a concentration (e.g., µg/mL). This MIC is then compared to interpretive criteria to determine susceptibility categories (S, I, or R)
  • Application: This method provides quantitative MIC values, offering more precision than disk diffusion. It is used for guiding antibiotic therapy, monitoring resistance, and can be useful for testing fastidious organisms. It is more expensive than disk diffusion, but provides a more accurate result than disk diffusion