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Microbiology

Critical Concentration

This section focuses on the critical concentration method, an approach used in antimicrobial susceptibility testing (AST) for Mycobacterium tuberculosis. It’s a simplified method compared to broth microdilution, but it still plays a role in determining drug resistance

General Principles

  • Qualitative Assessment: The critical concentration method is a qualitative or semi-quantitative AST method used primarily for Mycobacterium tuberculosis
  • Single Concentration: Unlike broth microdilution, the critical concentration method tests the organism’s ability to grow (or not grow) in the presence of a single, predetermined concentration of an antimicrobial agent
  • Critical Concentration: The critical concentration is a specific concentration of an antimicrobial agent that is expected to inhibit the growth of susceptible strains of M. tuberculosis but allow the growth of resistant strains
  • Simplified Method: Compared to broth microdilution, the critical concentration method is simpler to perform and interpret, making it suitable for resource-limited settings
  • Rapid Detection of Resistance: The critical concentration method can provide rapid detection of drug resistance in M. tuberculosis
  • Limitations: The critical concentration method does not provide quantitative MIC values and may not detect low-level resistance or resistance to multiple drugs

Procedure

  • Preparation of Media
    • Solid Media: The critical concentration method typically uses solid media, such as egg-based media (e.g., Löwenstein-Jensen) or agar-based media (e.g., Middlebrook 7H10)
    • Antimicrobial Agents: The antimicrobial agents are incorporated into the media at the predetermined critical concentrations
    • Quality Control: The media should be prepared according to established protocols and quality control procedures to ensure accurate results
  • Preparation of Inoculum
    • Standardization: Prepare a standardized inoculum of the M. tuberculosis isolate 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 inoculation
  • Inoculation of Media
    • Media with Antimicrobial Agent: Inoculate the solid media containing the antimicrobial agent at the critical concentration with the standardized inoculum
    • Control Media: Inoculate control media without the antimicrobial agent with the standardized inoculum to ensure viability and growth of the organism
  • Incubation
    • Temperature and Atmosphere: Incubate the inoculated media at the appropriate temperature and atmosphere for M. tuberculosis (e.g., 35-37°C in ambient air with 5-10% CO2)
    • Incubation Time: Incubate the media for the recommended incubation time, typically 3-8 weeks
  • Reading and Interpretation
    • Growth Assessment: After incubation, examine the media for the presence or absence of growth
    • Interpretation
      • Growth on Media with Antimicrobial Agent: Indicates resistance to the antimicrobial agent
      • No Growth on Media with Antimicrobial Agent: Indicates susceptibility to the antimicrobial agent
      • Growth on Control Media: Confirms viability and growth of the organism
    • Quality Control: Verify that the results for the quality control strains are within the acceptable range

Critical Concentrations for Common Anti-TB Drugs

  • Isoniazid (INH): 0.2 μg/mL or 1.0 μg/mL (depending on the medium and method)
  • Rifampin (RIF): 40 μg/mL
  • Ethambutol (EMB): 2.0 μg/mL or 5.0 μg/mL (depending on the medium and method)
  • Pyrazinamide (PZA): 100 μg/mL
  • Streptomycin (SM): 4.0 μg/mL or 10.0 μg/mL (depending on the medium and method)

Advantages

  • Simple to Perform: The critical concentration method is relatively simple to perform and requires minimal equipment
  • Cost-Effective: It is a cost-effective method, especially in resource-limited settings
  • Rapid Results: It provides relatively rapid results compared to other methods
  • Detection of Resistance: It can detect resistance to common anti-TB drugs

Limitations

  • Qualitative Results: It provides only qualitative or semi-quantitative results, without MIC values
  • Limited Scope: It tests only a limited number of antimicrobial agents
  • Detection of Low-Level Resistance: It may not detect low-level resistance or resistance to multiple drugs
  • Subjectivity: Interpretation of growth can be subjective
  • Media Preparation: Accurate media preparation is critical for reliable results
  • Does not replace molecular methods: Does not detect heteroresistance

Key Terms

  • Critical Concentration: A specific concentration of an antimicrobial agent that is expected to inhibit the growth of susceptible strains of M. tuberculosis but allow the growth of resistant strains
  • Antimicrobial Susceptibility Testing (AST): A laboratory test to determine the susceptibility of an organism to antimicrobial drugs
  • Minimum Inhibitory Concentration (MIC): The lowest concentration of an antimicrobial agent that inhibits the visible growth of the organism after a specified incubation period
  • Qualitative Test: A test that provides a result as either positive or negative, without quantifying the amount of substance present
  • Semi-Quantitative Test: A test that provides an approximate estimate of the amount of substance present
  • Standardized Inoculum: A suspension of microorganisms adjusted to a specific concentration
  • Egg-Based Media: Solid media containing egg as a nutrient source, such as Löwenstein-Jensen medium
  • Agar-Based Media: Solid media containing agar as a solidifying agent, such as Middlebrook 7H10 agar
  • Control Media: Media without antimicrobial agents used to ensure viability and growth of the organism
  • Quality Control: Measures taken to ensure the accuracy and reliability of laboratory tests
  • Inoculation: The process of introducing microorganisms into a culture medium
  • Incubation: Maintaining microorganisms under controlled conditions to promote growth
  • Resistance: The ability of an organism to grow in the presence of an antimicrobial agent at a concentration that would normally inhibit growth
  • Susceptibility: The inability of an organism to grow in the presence of an antimicrobial agent at a concentration that is normally used to inhibit growth
  • Löwenstein-Jensen (LJ) Medium: An egg-based medium commonly used for the cultivation of mycobacteria
  • Middlebrook 7H10 Agar: An agar-based medium used for the cultivation of mycobacteria
  • Quality Control Strains: Strains of microorganisms with known susceptibility patterns used to verify the accuracy of antimicrobial susceptibility tests
  • Turbidity: Cloudiness or haziness of a liquid caused by the presence of suspended particles
  • CO2: Carbon dioxide
  • μg/mL: Micrograms per milliliter, a unit of concentration
  • Qualitative Results: Results that are expressed as either positive or negative
  • Heteroresistance: The presence of both susceptible and resistant subpopulations within a bacterial population