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Microbiology

Mycobacteria & Fungi

Mycobacteria and fungi present unique challenges in the clinical microbiology laboratory due to their slow growth rates, specific nutritional requirements, and potential for environmental contamination. Specialized media are crucial for their isolation, identification, and characterization

Specialized Media for Mycobacteria and Fungi: Meeting Unique Growth Requirements

  • Why are Mycobacteria and Fungi Challenging to Culture?
    • Slow Growth Rates: Many mycobacteria and fungi grow slowly, requiring prolonged incubation periods
    • Specific Nutritional Requirements: Mycobacteria and fungi often require specific nutrients and growth factors not found in routine media
    • Environmental Contamination: Mycobacteria and fungi are ubiquitous in the environment, increasing the risk of contamination
    • Cell Wall Structure: The complex cell wall structure of mycobacteria and fungi can make them resistant to certain antimicrobial agents
  • General Strategies for Culturing Mycobacteria and Fungi
    • Specimen Processing: Digestion and decontamination of specimens to eliminate non-target organisms
    • Selective Media: Use of media containing selective agents to inhibit the growth of bacteria and other fungi
    • Prolonged Incubation: Incubation for several weeks to allow for slow-growing organisms to grow
    • Atmospheric Control: Providing the appropriate atmospheric conditions (e.g., aerobic, microaerophilic)
    • Temperature Control: Maintaining the optimal temperature for growth
    • Humidification: Providing adequate humidity to prevent desiccation

Specialized Media for Mycobacteria

Egg-Based Media

  • Principle: Egg-based media contain egg yolk, which provides a rich source of nutrients and lipids for mycobacterial growth
  • Examples
    • Lowenstein-Jensen (LJ) Medium: Contains egg, glycerol, mineral salts, and malachite green
    • Petragnani Medium: Contains egg, potato flour, and malachite green
  • Malachite Green: Inhibits the growth of most bacteria other than mycobacteria
  • Applications
    • Isolation of Mycobacterium tuberculosis from respiratory specimens
    • Isolation of other Mycobacterium species from clinical specimens
  • Key Considerations
    • LJ and Petragnani media should be incubated at 35-37°C in a humidified atmosphere with 5-10% CO2
    • Cultures should be incubated for up to 8 weeks to allow for slow-growing Mycobacterium species to grow
    • Colonies of M. tuberculosis typically appear as rough, buff-colored, and slow-growing
    • Confirmation of Mycobacterium identification requires biochemical testing, molecular methods, or lipid analysis

Agar-Based Media

  • Principle: Agar-based media contain agar as a solidifying agent and are enriched with various nutrients and growth factors
  • Examples
    • Middlebrook 7H10 Agar: Contains Middlebrook 7H10 broth, oleic acid-albumin-dextrose-catalase (OADC) enrichment, and glycerol
    • Middlebrook 7H11 Agar: Similar to 7H10, but also contains casein hydrolysate and malachite green
  • OADC Enrichment: Provides lipids, albumin, catalase, and dextrose to support mycobacterial growth
  • Applications
    • Isolation of Mycobacterium tuberculosis from respiratory specimens
    • Isolation of other Mycobacterium species from clinical specimens
    • Antimicrobial susceptibility testing of Mycobacterium tuberculosis
  • Key Considerations
    • Middlebrook 7H10 and 7H11 agars should be incubated at 35-37°C in a humidified atmosphere with 5-10% CO2
    • Cultures should be incubated for up to 8 weeks to allow for slow-growing Mycobacterium species to grow
    • Colonies of M. tuberculosis typically appear as small, dry, and granular
    • Confirmation of Mycobacterium identification requires biochemical testing, molecular methods, or lipid analysis

Liquid Media

  • Principle: Liquid media provide a more rapid and sensitive method for detecting mycobacteria
  • Examples
    • Middlebrook 7H9 Broth: Contains Middlebrook 7H9 broth, OADC enrichment, and glycerol
    • Bactec MGIT (Mycobacteria Growth Indicator Tube): Contains modified Middlebrook 7H9 broth with an oxygen sensor that fluoresces in the presence of mycobacterial growth
  • Automated Systems: Bactec MGIT is used in automated systems that continuously monitor for mycobacterial growth
  • Applications
    • Rapid detection of Mycobacterium tuberculosis from respiratory specimens
    • Antimicrobial susceptibility testing of Mycobacterium tuberculosis
  • Key Considerations
    • Liquid media should be incubated at 35-37°C in a humidified atmosphere with 5-10% CO2
    • Cultures should be monitored regularly for growth
    • Positive cultures should be confirmed by smear microscopy and identification methods

Selective Media for Mycobacteria

  • Principle: Selective media contain antimicrobial agents that inhibit the growth of bacteria and fungi, allowing for the selective isolation of mycobacteria
  • Examples
    • LJ Medium with Antimicrobials: LJ medium supplemented with antibiotics such as polymyxin B, amphotericin B, and nalidixic acid
    • Middlebrook 7H11 Agar with Antimicrobials: Middlebrook 7H11 agar supplemented with antibiotics
  • Applications
    • Isolation of Mycobacterium species from specimens with high levels of bacterial or fungal contamination
  • Key Considerations
    • Selective media should be used with caution, as they may inhibit the growth of some Mycobacterium species
    • Cultures should be monitored carefully for growth

Specialized Media for Fungi

Sabouraud Dextrose Agar (SDA)

  • Principle: SDA contains a high concentration of dextrose (glucose) and a low pH, which inhibits the growth of bacteria and selects for fungi
  • Composition: Contains peptone and dextrose
  • Applications
    • Isolation of fungi from clinical specimens
    • Maintenance of stock cultures of fungi
  • Key Considerations
    • SDA should be incubated at 25-30°C in a humidified atmosphere
    • Cultures should be monitored regularly for growth
    • Colony morphology and microscopic examination are used to identify fungi

Brain Heart Infusion (BHI) Agar

  • Principle: BHI agar is a nutrient-rich medium that supports the growth of a wide range of fungi
  • Composition: Contains brain heart infusion, peptone, and dextrose
  • Applications
    • Isolation of fungi from clinical specimens
    • Growth of fastidious fungi
  • Key Considerations
    • BHI agar should be incubated at 25-30°C in a humidified atmosphere
    • Cultures should be monitored regularly for growth
    • Colony morphology and microscopic examination are used to identify fungi

Inhibitory Mold Agar (IMA)

  • Principle: IMA contains chloramphenicol, which inhibits the growth of bacteria, allowing for the selective isolation of fungi
  • Composition: Contains BHI agar with chloramphenicol
  • Applications
    • Selective isolation of fungi from specimens with bacterial contamination
  • Key Considerations
    • IMA should be incubated at 25-30°C in a humidified atmosphere
    • Cultures should be monitored regularly for growth
    • Colony morphology and microscopic examination are used to identify fungi

Dermatophyte Test Medium (DTM)

  • Principle: DTM contains cycloheximide and gentamicin to inhibit the growth of bacteria and saprophytic fungi, and a pH indicator (phenol red) to detect dermatophytes
  • Composition: Contains peptone, dextrose, cycloheximide, gentamicin, and phenol red
  • Applications
    • Selective isolation and identification of dermatophytes from skin, hair, and nail specimens
  • Rationale
    • Dermatophytes produce alkaline metabolites, which turn the pH indicator red
  • Key Considerations
    • DTM should be incubated at 25-30°C in a humidified atmosphere
    • Cultures should be monitored regularly for color change and growth
    • Microscopic examination of fungal structures is used to confirm the identification of dermatophytes

Potato Dextrose Agar (PDA)

  • Principle: PDA contains potato infusion and dextrose, which promote sporulation and pigment production in fungi
  • Composition: Contains potato infusion and dextrose
  • Applications
    • Enhancing sporulation and pigment production in fungi
    • Identification of fungi based on colony morphology
  • Key Considerations
    • PDA should be incubated at 25-30°C in a humidified atmosphere
    • Cultures should be monitored regularly for growth and pigment production
    • Microscopic examination of fungal structures is used to confirm the identification of fungi

Key Considerations and Best Practices

  • Specimen Collection: Collect specimens properly to minimize contamination and ensure adequate sample volume
  • Transport: Transport specimens promptly to the laboratory in appropriate transport media
  • Specimen Processing: Digestion and decontamination of specimens to eliminate non-target organisms
  • Media Selection: Choose the appropriate specialized media based on the suspected pathogens and the specimen type
  • Incubation Conditions: Follow the recommended incubation conditions for each medium, including temperature, atmosphere, and duration
  • Quality Control: Perform quality control testing on all media to ensure proper performance
  • Identification: Use appropriate identification methods to confirm the identity of isolated bacteria
  • Documentation: Document all media selection, incubation conditions, and identification results

Key Terms

  • Mycobacteria: A genus of bacteria that includes Mycobacterium tuberculosis and other species that can cause disease
  • Fungi: A kingdom of eukaryotic organisms that includes yeasts, molds, and mushrooms
  • Fastidious: Having complex nutritional requirements
  • Specialized Media: Culture media formulated to support the growth of specific microorganisms
  • Selective Media: Media that inhibit the growth of certain microorganisms while allowing others to grow
  • Enriched Media: Media that contain specific nutrients or growth factors required by fastidious microorganisms
  • Quality Control: A set of procedures designed to ensure the accuracy and reliability of laboratory test results
  • Standard Operating Procedure (SOP): A detailed written instruction to achieve uniformity of the performance of a specific function