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Microbiology

Culture Methods

This section covers culture methods used for MRSA/MSSA, VRE, ESBL/CRE screening. This is where we get to see the bacteria “grow” and give us clues about their identity and resistance

MRSA/MSSA Screening

  • Selective Media
    • Chromogenic Media: These media contain chromogenic substrates that are cleaved by bacterial enzymes, producing distinct colored colonies. For MRSA, the media often contain an antibiotic (e.g., oxacillin or cefoxitin) and a chromogen that turns the S. aureus colonies a specific color (e.g., pink, mauve, or blue) if the bacteria are resistant
    • Mannitol Salt Agar (MSA): This is a differential and selective media. It contains a high concentration of salt, which inhibits most bacteria except staphylococci. S. aureus ferments mannitol, producing acid and turning the pH indicator (phenol red) yellow
  • Non-Selective Media
    • Blood Agar Plate (BAP): All swabs are plated on BAP. This allows for the growth of all bacteria
  • Incubation: Incubate at 35-37°C in ambient air (no added CO2) for 24-48 hours
  • Identification
    • Colony Morphology: S. aureus typically appears as large, round, creamy, or golden colonies on BAP
    • Gram Stain: Gram-positive cocci in clusters
    • Coagulase Test: This is the gold standard for differentiating S. aureus from other staphylococci. Coagulase-positive indicates S. aureus
    • Oxacillin or Cefoxitin Disk Diffusion: This is a standard method to determine methicillin resistance
    • Latex Agglutination: Latex agglutination tests are available to detect the presence of PBP2a (the protein encoded by the mecA gene) or to confirm S. aureus

VRE Screening

  • Selective Media
    • Bile Esculin Agar (BEA) with Vancomycin: BEA is used because it selects for enterococci. The addition of vancomycin inhibits vancomycin-susceptible organisms
    • Chromogenic Media: Similar to MRSA, chromogenic media can be used. The media contains vancomycin and a chromogen that helps identify enterococci
  • Incubation: Incubate at 35-37°C in ambient air for 24-48 hours
  • Identification
    • Colony Morphology: Enterococci typically appear as small, white or gray colonies on BAP or BEA
    • Gram Stain: Gram-positive cocci in pairs or short chains
    • Catalase Test: Negative (enterococci are catalase-negative)
    • Vancomycin Susceptibility Testing: Performed using disk diffusion or automated methods to confirm vancomycin resistance

ESBL/CRE Screening

  • Selective Media
    • Chromogenic Media: These media contain a chromogen and an antibiotic to select for ESBL- or carbapenem-resistant organisms
    • MacConkey Agar: This is a differential media, allowing for the growth of Gram-negative bacteria while inhibiting Gram-positive organisms
  • Incubation: Incubate at 35-37°C in ambient air for 24-48 hours
  • Identification
    • Colony Morphology: The morphology depends on the specific organism
    • Gram Stain: Gram-negative bacilli
    • Identification and Susceptibility Testing
      • Automated Systems: Automated identification and susceptibility testing systems (e.g., Vitek, Phoenix) are commonly used
      • Modified Kirby-Bauer Disk Diffusion: Used to test for ESBL production
      • Carbapenem Susceptibility Testing: Disk diffusion or MIC determination is performed
      • Phenotypic Tests
        • ESBL Detection: Double-disk synergy test (using a cephalosporin and clavulanic acid), or other phenotypic tests
        • Carbapenemase Detection: Modified Hodge test, Carba NP test, or other phenotypic tests

Key Terms with Definitions

  • Selective Media: Culture media designed to inhibit the growth of some organisms while allowing the growth of others, often containing antibiotics or other inhibitory agents
  • Differential Media: Culture media that allows the growth of multiple organisms but differentiates them based on their biochemical properties, often by the use of pH indicators or other reagents
  • Chromogenic Media: Culture media that contains chromogenic substrates that are cleaved by bacterial enzymes, producing distinct colored colonies
  • Colony Morphology: The visible characteristics of bacterial colonies on an agar plate, including size, shape, color, texture, and edge
  • Gram Stain: A differential staining method used to classify bacteria based on their cell wall structure, resulting in either Gram-positive (purple) or Gram-negative (pink/red) staining
  • Catalase Test: A biochemical test to detect the presence of the enzyme catalase, which converts hydrogen peroxide to water and oxygen. Used to differentiate between staphylococci (catalase-positive) and streptococci (catalase-negative)
  • Coagulase Test: A biochemical test used to differentiate Staphylococcus aureus (coagulase-positive) from other staphylococci (coagulase-negative)
  • Disk Diffusion: A susceptibility testing method where antibiotic-impregnated disks are placed on an agar plate inoculated with the test organism, and the zone of inhibition around the disk indicates susceptibility
  • Minimum Inhibitory Concentration (MIC): The lowest concentration of an antimicrobial agent that inhibits the visible growth of a microorganism
  • Zone of Inhibition: The clear area around an antibiotic disk on an agar plate, where bacterial growth is inhibited due to the antibiotic’s effect