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Microbiology

Gradient Diffusion

This method is a variation of the disk diffusion method, offering a more precise and quantitative approach to antimicrobial susceptibility testing

Method

  • Preparation
    1. Inoculum Preparation: A standardized bacterial suspension (typically a 0.5 McFarland standard) is prepared, similar to disk diffusion
    2. Test Strip Application: A commercially available test strip is applied to the surface of a Mueller-Hinton agar plate. The strip contains a predefined gradient of an antimicrobial agent, with a high concentration at one end and a low concentration at the other
  • Procedure
    1. Inoculation: The standardized bacterial suspension is evenly spread over the surface of the Mueller-Hinton agar plate
    2. Strip Placement: The test strip is placed on the agar surface. The strip is usually applied using sterile forceps
    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, an elliptical zone of inhibition forms where the antibiotic has inhibited bacterial growth
    2. MIC Determination: The MIC value is read directly from the test strip at the point where the elliptical zone of inhibition intersects the strip. The MIC value is printed on the strip, usually in µg/mL

Theory

  • Diffusion and Concentration Gradient: The test strip contains a pre-defined, continuous gradient of an antibiotic. As the antibiotic diffuses into the agar, it creates a concentration gradient around the strip
  • Growth Inhibition and MIC: The antibiotic inhibits bacterial growth at concentrations above the MIC. The point where the growth inhibition zone intersects the strip represents the MIC value
  • Quantitative Result: Unlike disk diffusion, gradient diffusion provides a quantitative MIC value, similar to microbroth dilution

Interpretation

  • MIC Value: The MIC value is read directly from the test strip. The value is expressed as a concentration (e.g., µg/mL)
  • Susceptibility Categories: The MIC value is interpreted using established interpretive criteria provided by CLSI (or other guidelines). The 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
    • Resistant (R): The antibiotic is unlikely to be effective at any achievable dosage
  • Reporting: The MIC and the corresponding susceptibility category (S, I, or R) are reported to the clinician

Application

  • Clinical Use
    • Guiding Antibiotic Therapy: The MIC value helps guide clinicians in selecting the most effective antibiotic for treating bacterial infections
    • Monitoring Antibiotic Resistance: The method aids in tracking the emergence and spread of antibiotic resistance
    • Testing Fastidious Organisms: Gradient diffusion can be used to test fastidious organisms, such as anaerobes, that may not grow well with other methods
  • Advantages of Gradient Diffusion
    • Quantitative Results: Provides a precise MIC value, allowing for more informed clinical decisions
    • Ease of Use: The method is relatively easy to perform and interpret
    • Versatility: Can be used to test a wide range of antibiotics and bacterial species
    • Higher Accuracy: Provides more accurate results compared to disk diffusion, especially for intermediate susceptibility
    • Testing of Anaerobes and Fastidious Organisms: It is particularly useful for testing anaerobes and other fastidious organisms, as the results are often clearer than with disk diffusion
  • Disadvantages of Gradient Diffusion
    • Cost: The test strips are more expensive than antibiotic disks
    • Limited Antibiotic Selection: Not all antibiotics are available in gradient diffusion format
    • Potential for Variability: The interpretation of the MIC can be subjective, especially when the zone of inhibition is not clear-cut

Important Considerations

  • Quality Control: Strict quality control measures are essential for accurate results. This includes testing with control organisms (e.g., Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922) to verify the performance of the test system
  • Media: Mueller-Hinton agar is the standard medium for gradient diffusion testing
  • Inoculum: The inoculum density is critical. Too many or too few bacteria can affect the results
  • Strip Storage: Store the test strips according to the manufacturer’s instructions (usually refrigerated)
  • Application Technique: Apply the test strips correctly according to the manufacturer’s instructions
  • Incubation Conditions: Maintain the correct incubation temperature and atmosphere
  • Reading and Interpretation: Read the MIC value carefully, following the manufacturer’s instructions and CLSI guidelines

Key Terms

  • Gradient Diffusion: A method of antimicrobial susceptibility testing that uses test strips with a pre-defined antibiotic gradient
  • Test Strip: A commercially available strip containing a gradient of an antimicrobial agent
  • MIC (Minimum Inhibitory Concentration): The lowest concentration of an antimicrobial agent that completely inhibits the visible growth of a microorganism after incubation. This is the most important result of the test
  • Zone of Inhibition: The area around the test strip where bacterial growth is inhibited
  • Mueller-Hinton Agar: The standard agar medium used for gradient diffusion testing
  • 0.5 McFarland Standard: A turbidity standard used to prepare the bacterial inoculum
  • 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
  • Inoculation: The process of spreading the bacterial suspension onto the agar plate