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Microbiology

Disk Diffusion

This method is a widely used for antimicrobial susceptibility testing and is a relatively straightforward technique for determining if a bacterium is susceptible or resistant to a particular antibiotic

Method

  • Preparation
    1. Inoculum Preparation: A pure culture of the bacterium is grown, and a standardized suspension (typically a 0.5 McFarland standard) is prepared
    2. Media Preparation: A Mueller-Hinton agar plate is used. The agar surface should be free of moisture
    3. Disk Application: Antibiotic-impregnated disks, each containing a specific concentration of a single antibiotic, are placed on the agar surface
  • Procedure
    1. Inoculation: The standardized bacterial suspension is evenly spread over the surface of the Mueller-Hinton agar plate
    2. Disk Placement: Antibiotic disks are applied to the inoculated agar surface. A disk dispenser is commonly used to ensure proper spacing
    3. Incubation: The plates are incubated at 35°C in ambient air (unless otherwise specified) for 16-24 hours
  • Reading
    1. Zone of Inhibition: After incubation, the plates are examined for zones of inhibition (clear areas around the disks) where bacterial growth has been inhibited by the antibiotic
    2. Zone Diameter Measurement: The diameter of each zone of inhibition is measured in millimeters (mm) using a ruler or a zone reader
    3. Interpretation: The zone diameter is compared to established interpretive criteria (provided by CLSI or other guidelines) to determine if the bacterium is susceptible (S), intermediate (I), or resistant (R) to the antibiotic

Theory

  • Diffusion: Antibiotics diffuse radially outward from the paper disk into the agar medium
  • Growth Inhibition: As the antibiotic diffuses, it inhibits the growth of susceptible bacteria in the surrounding agar
  • Zone Diameter and MIC Correlation: The size of the zone of inhibition is inversely proportional to the MIC of the antibiotic for the bacterium. A larger zone indicates a lower MIC (greater susceptibility), and a smaller zone indicates a higher MIC (reduced susceptibility or resistance)
  • Antibiotic Properties: The effectiveness of the disk diffusion method depends on several factors, including:
    • Antibiotic diffusion rate in the agar
    • Antibiotic stability
    • Antibiotic potency
    • Bacterial growth rate

Interpretation

  • Zone Diameter and Susceptibility Categories
    • The measured zone diameter is compared to the interpretive criteria provided by CLSI (or other guidelines). These criteria are based on the correlation between zone diameters and MIC values
    • The interpretive criteria categorize the bacterium as:
      • Susceptible (S): The antibiotic is likely to be effective at the standard dosage
      • Intermediate (I): The antibiotic may be effective at a higher dosage or in body sites where the antibiotic concentrates. The result is often considered a “gray zone”
      • Resistant (R): The antibiotic is unlikely to be effective at any achievable dosage
  • Reporting: The zone diameter (in mm) and the corresponding susceptibility category (S, I, or R) are reported to the clinician

Application

  • Clinical Use
    • Guiding Antibiotic Therapy: The primary application is to guide clinicians in selecting the most appropriate antibiotic for treating bacterial infections
    • Monitoring Antibiotic Resistance: Repeated susceptibility testing helps track the emergence and spread of antibiotic resistance in a population
  • Advantages of Disk Diffusion
    • Simplicity and Cost-Effectiveness: The method is relatively simple to perform and requires minimal equipment, making it cost-effective
    • Versatility: Can be used to test a wide range of antibiotics and bacterial species
    • Widely Used: Is a well-established method with extensive data available for interpretation
  • Disadvantages of Disk Diffusion
    • Qualitative or Semi-Quantitative: Provides only qualitative or semi-quantitative results (susceptible, intermediate, resistant), unlike the quantitative MIC results obtained with microbroth dilution
    • Less Precise: The zone diameter measurement can be subject to some variability
    • Not Suitable for All Organisms: May not be suitable for testing fastidious organisms or organisms that grow poorly on Mueller-Hinton agar

Important Considerations

  • Quality Control: Strict quality control measures are essential for accurate results. This includes testing with control organisms (e.g., Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922) to verify the performance of the test system
  • Media: Mueller-Hinton agar is the standard medium for disk diffusion testing. The agar depth and pH must be appropriate
  • Inoculum: The inoculum density is critical. Too many or too few bacteria can affect the zone diameters
  • Disk Quality: Use only commercially prepared, quality-controlled antibiotic disks
  • Incubation Conditions: Maintain the correct incubation temperature and atmosphere
  • Zone Diameter Measurement: Measure the zone diameters carefully, following established guidelines
  • Interpretive Criteria: Always refer to the most recent CLSI guidelines for up-to-date interpretive criteria

Key Terms

  • Zone of Inhibition: The clear area around an antibiotic disk where bacterial growth is inhibited
  • Disk Diffusion: The method of antimicrobial susceptibility testing that uses antibiotic-impregnated disks on agar plates
  • Mueller-Hinton Agar: The standard agar medium used for disk diffusion testing
  • 0.5 McFarland Standard: A turbidity standard used to prepare the bacterial inoculum
  • Zone Diameter: The diameter of the zone of inhibition, measured in millimeters (mm)
  • Susceptible (S): A category indicating that the microorganism is inhibited by the antimicrobial agent at concentrations achievable at the site of infection using the normal dosage
  • Intermediate (I): A category indicating that the microorganism may be inhibited by the antimicrobial agent at a higher concentration, or when the agent is concentrated at the site of infection (e.g., urine). It also suggests that the outcome may be unpredictable
  • Resistant (R): A category indicating that the microorganism is not inhibited by the antimicrobial agent at concentrations achievable at the site of infection using the normal dosage. Treatment with this agent is likely to fail
  • CLSI (Clinical and Laboratory Standards Institute): An organization that provides standardized methods and interpretive criteria for antimicrobial susceptibility testing. They publish guidelines that are widely adopted by clinical laboratories
  • Antibiotic Disk: A paper disk impregnated with a specific concentration of an antibiotic
  • Inoculation: The process of spreading the bacterial suspension onto the agar plate