Skip to main content

Microbiology

Microbroth Dilution

This method is all about determining the minimum inhibitory concentration (MIC) of an antibiotic, which is the lowest concentration that prevents visible growth of a bacterium. This section covers the method, theory, interpretation, and application of microbroth dilution:

Method

  • Preparation
    • Inoculum Preparation: The process begins by preparing a standardized bacterial inoculum. This typically involves growing the bacteria in a broth medium (e.g., Mueller-Hinton broth) to a specific turbidity, often equivalent to a 0.5 McFarland standard. This standardization is crucial for reproducible results
    • Antibiotic Dilutions: Serial dilutions of the antimicrobial agents are prepared in microtiter plates. These plates have multiple wells, and each well contains a different concentration of the antibiotic. The concentrations typically range from high to low, covering a spectrum of potential inhibitory levels
  • Procedure
    1. Plate Setup: A microtiter plate (usually 96-well) is used
    2. Inoculation: The standardized bacterial suspension is added to each well of the microtiter plate, along with the diluted antibiotics
    3. Incubation: The microtiter plate is incubated under appropriate conditions (e.g., 35°C, aerobic atmosphere) for a specified duration (typically 18-24 hours)
  • Reading
    1. Visual Observation: After incubation, the plates are examined visually for bacterial growth. Growth is typically indicated by turbidity (cloudiness) in the wells
    2. MIC Determination: The MIC is the lowest concentration of the antibiotic that completely inhibits visible bacterial growth. This is the well where the broth remains clear

Theory

  • The Principle: Microbroth dilution is based on the principle of exposing bacteria to a range of antibiotic concentrations. The antibiotic inhibits bacterial growth by interfering with essential cellular processes
  • Antibiotic Mechanisms of Action: Different antibiotics work by targeting different bacterial structures or processes, such as:
    • Cell Wall Synthesis: Beta-lactams (penicillins, cephalosporins), vancomycin
    • Protein Synthesis: Aminoglycosides, macrolides, tetracyclines, chloramphenicol
    • DNA Synthesis: Quinolones, sulfonamides, trimethoprim
    • RNA Synthesis: Rifampin
  • Bacterial Resistance: Bacteria can develop resistance to antibiotics through various mechanisms, including:
    • Enzymatic inactivation: e.g., beta-lactamases
    • Target modification: e.g., altered penicillin-binding proteins (PBPs)
    • Reduced permeability: e.g., efflux pumps
    • Target overproduction: e.g., overproduction of dihydropteroate synthase (DHPS)

Interpretation

  • MIC Values: The MIC is expressed as a concentration (e.g., µg/mL). This value is crucial for guiding antibiotic selection and dosage
  • Susceptibility Categories: The MIC value is interpreted using established interpretive criteria, which are typically provided by organizations like the Clinical and Laboratory Standards Institute (CLSI). These criteria categorize the bacteria as:
    • Susceptible (S): The antibiotic is likely to be effective at the standard dose
    • Intermediate (I): The antibiotic may be effective at a higher dose or in certain body sites where the antibiotic concentrates
    • Resistant (R): The antibiotic is unlikely to be effective at any achievable dose
  • Reporting: The MIC 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 effective antibiotic for treating a specific infection
    • Monitoring Antibiotic Resistance: Repeated susceptibility testing helps track the emergence and spread of antibiotic resistance in a population
    • Dosage Optimization: The MIC can be used to calculate the appropriate antibiotic dosage to achieve effective concentrations at the site of infection
  • Advantages of Microbroth Dilution
    • Quantitative Results: Provides a precise MIC value, allowing for more informed clinical decisions
    • Standardization: The method is highly standardized, ensuring reproducibility and comparability of results
    • Versatility: Can be used to test a wide range of antibiotics and bacterial species
  • Disadvantages of Microbroth Dilution
    • Labor-Intensive: Requires careful technique and is more time-consuming than some other methods
    • Equipment: Requires specialized equipment, such as microtiter plates and automated readers (for some applications)
  • Automation: Many clinical microbiology laboratories use automated systems that perform microbroth dilution, significantly increasing throughput and reducing manual labor

Important Considerations

  • Quality Control: Strict quality control measures are essential to ensure the accuracy and reliability of microbroth dilution 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: The choice of growth media can influence the MIC results. Mueller-Hinton broth is the most commonly used medium for routine susceptibility testing
  • Inoculum Density: Maintaining the correct inoculum density is critical. Too many or too few bacteria can lead to inaccurate MIC values
  • Interpretive Criteria: Always refer to the most recent CLSI guidelines for the most up-to-date interpretive criteria. These criteria are updated periodically to reflect changes in antibiotic resistance patterns and clinical practice

Key Terms

  • 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
  • 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
  • Inoculum:* The standardized suspension of bacteria used in the susceptibility test. The inoculum density (usually a 0.5 McFarland standard) is critical for accurate results
  • Mueller-Hinton Broth:* The standard broth medium used for antimicrobial susceptibility testing. It’s chosen for its ability to support the growth of most non-fastidious bacteria and its well-defined composition
  • Microtiter Plate:* A plastic plate with multiple small wells (typically 96 wells) used to perform the microbroth dilution test. Each well contains a different concentration of the antibiotic
  • Beta-Lactamase:* An enzyme produced by some bacteria that inactivates beta-lactam antibiotics (e.g., penicillins, cephalosporins) by breaking the beta-lactam ring
  • 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 Resistance:* The ability of a microorganism to survive and multiply in the presence of an antibiotic. This can be due to various mechanisms, such as enzymatic inactivation, target modification, or reduced permeability