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Microbiology

Broth Microdilution

This section will focus on broth microdilution, a key method for determining the antimicrobial susceptibility of Mycobacteria and Nocardia species. We’ll explore the principles, procedure, advantages, and limitations of this technique

General Principles

  • Purpose: Broth microdilution is a quantitative method used to determine the minimum inhibitory concentration (MIC) of an antimicrobial agent against a specific organism
  • MIC: The MIC is defined as the lowest concentration of an antimicrobial agent that inhibits the visible growth of the organism after a specified incubation period
  • Quantitative Results: Broth microdilution provides quantitative MIC values, which can be used to categorize organisms as susceptible, intermediate, or resistant based on established breakpoints
  • Standardized Method: Standardized broth microdilution methods are available from organizations such as the Clinical and Laboratory Standards Institute (CLSI)
  • Clinical Significance: MIC values obtained from broth microdilution testing can be used to guide antimicrobial therapy and predict clinical outcomes

Procedure

  • Preparation of Antimicrobial Agents
    • Stock Solutions: Prepare stock solutions of antimicrobial agents in appropriate solvents according to manufacturer’s instructions
    • Serial Dilutions: Prepare serial two-fold dilutions of the antimicrobial agents in a suitable broth medium (e.g., cation-adjusted Mueller-Hinton broth, Middlebrook 7H9 broth)
  • Preparation of Inoculum
    • Standardization: Prepare a standardized inoculum of the test organism in a suitable broth medium. The inoculum density is typically adjusted to a specific turbidity (e.g., 0.5 McFarland standard)
    • Dilution: Dilute the standardized inoculum to the appropriate concentration for testing
  • Microdilution Plate Setup
    • Microdilution Plates: Use sterile, disposable microdilution plates with multiple wells. Each well contains a specific concentration of the antimicrobial agent
    • Inoculation: Inoculate each well of the microdilution plate with the standardized inoculum
    • Controls: Include appropriate controls, such as:
      • Growth Control: Wells containing broth medium and inoculum but no antimicrobial agent
      • Sterility Control: Wells containing broth medium only to check for contamination
      • Quality Control Strains: Wells inoculated with quality control strains of known susceptibility to verify the accuracy of the test
  • Incubation
    • Temperature and Atmosphere: Incubate the microdilution plates at the appropriate temperature and atmosphere for the test organism (e.g., 35°C in ambient air for most bacteria, 30°C for M. marinum)
    • Incubation Time: Incubate the plates for the recommended incubation time, which varies depending on the organism (e.g., 18-24 hours for most bacteria, several days to weeks for Mycobacteria)
  • Reading and Interpretation
    • Visual Inspection: After incubation, examine the microdilution plates visually to determine the MIC. The MIC is the lowest concentration of the antimicrobial agent that inhibits visible growth of the organism
    • Turbidity: Wells with growth will appear turbid, while wells without growth will appear clear
    • Quality Control: Verify that the results for the quality control strains are within the acceptable range
    • Interpretation: Interpret the MIC values based on established breakpoints to categorize the organism as susceptible, intermediate, or resistant

Advantages

  • Quantitative Results: Provides quantitative MIC values, which are more informative than qualitative results (e.g., disk diffusion)
  • Automation: Can be automated, allowing for high-throughput testing
  • Cost-Effective: Relatively cost-effective compared to other methods, especially when automated
  • Versatile: Can be used to test a wide range of antimicrobial agents and organisms
  • Standardized: Standardized methods are available from CLSI and other organizations

Limitations

  • Labor-Intensive: Manual broth microdilution can be labor-intensive
  • Subjectivity: Visual reading of the microdilution plates can be subjective
  • Media and Reagents: Requires specialized media and reagents
  • Time-Consuming: Can be time-consuming, especially for slow-growing organisms
  • Interpretation Challenges: Interpretation of MIC values can be challenging, especially for organisms with unusual resistance mechanisms or limited breakpoint data
  • Biofilms: Broth microdilution may not accurately predict the activity of antimicrobial agents against organisms growing in biofilms

Key Terms

  • Broth Microdilution: A quantitative method for determining the minimum inhibitory concentration (MIC) of an antimicrobial agent against a specific organism
  • MIC (Minimum Inhibitory Concentration): The lowest concentration of an antimicrobial agent that inhibits the visible growth of the organism after a specified incubation period
  • Breakpoint: A concentration of an antimicrobial agent used to categorize organisms as susceptible, intermediate, or resistant based on MIC values
  • Susceptible: An organism is considered susceptible to an antimicrobial agent if the MIC is below the breakpoint
  • Intermediate: An organism is considered intermediate to an antimicrobial agent if the MIC is at or near the breakpoint
  • Resistant: An organism is considered resistant to an antimicrobial agent if the MIC is above the breakpoint
  • Inoculum: A suspension of microorganisms used to start a culture
  • Turbidity: Cloudiness or haziness of a liquid caused by the presence of suspended particles
  • Quality Control: Measures taken to ensure the accuracy and reliability of laboratory tests
  • Control Strains: Strains of microorganisms with known susceptibility patterns used to verify the accuracy of antimicrobial susceptibility tests
  • Automation: The use of automated instruments to perform laboratory tests
  • High-Throughput Testing: The ability to perform a large number of tests in a short period of time
  • Cation-Adjusted Mueller-Hinton Broth: A standardized broth medium used for antimicrobial susceptibility testing
  • Middlebrook 7H9 Broth: A broth medium used for the cultivation and antimicrobial susceptibility testing of Mycobacteria
  • Serial Dilutions: A series of dilutions in which the concentration of a substance is decreased by the same factor in each step
  • :** McFarland Standard:** A suspension of barium sulfate used to standardize the inoculum density for antimicrobial susceptibility testing
  • Biofilm: A community of microorganisms attached to a surface, enclosed in a self-produced matrix
  • CLSI (Clinical and Laboratory Standards Institute): A non-profit organization that develops standards and guidelines for laboratory testing
  • NCCLS (National Committee for Clinical Laboratory Standards): The former name of CLSI
  • Antimicrobial Agent: A substance that kills or inhibits the growth of microorganisms
  • Sterility Control: A control used to check for contamination in media and reagents
  • Growth Control: A control used to ensure that the organism is growing properly in the absence of antimicrobial agents
  • Visual Inspection: Examination of the microdilution plates with the naked eye to determine the MIC
  • Quality Control Strains: Strains of microorganisms with known susceptibility patterns used to verify the accuracy of antimicrobial susceptibility tests
  • Solvent: A liquid used to dissolve a substance