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Microbiology

Identification Methods

This section encompasses a range of methods used in the clinical microbiology laboratory to identify bacteria, from the initial observations to advanced molecular techniques. Each method contributes to a comprehensive identification process, and their results are often correlated to reach a definitive conclusion

Initial Assessment & Foundation

  • Colony Morphology
    • Theory: Visual characteristics of bacterial colonies on solid media (e.g., agar plates). Influenced by agar type, nutrients, temperature, and organism characteristics
    • Interpretation: Observing and describing size, shape, elevation, margin, surface, opacity, color, odor, and hemolysis. This provides initial clues and guides further testing
    • Application: A critical first step. Used to differentiate organisms, guide selection of other tests, and correlate with Gram stain
  • Gram Stain
    • Theory: Differential staining technique that classifies bacteria based on cell wall structure (Gram-positive vs. Gram-negative)
    • Interpretation: Observation of cell shape (cocci, bacilli, spirilla), arrangement (clusters, chains, pairs), and staining properties (purple for Gram-positive, pink/red for Gram-negative)
    • Application: Provides immediate information about the organism’s morphology and Gram reaction, guiding subsequent testing. Always performed before any other identification test

Presumptive Identification Methods

These methods offer rapid, preliminary identification results

  • Rapid Tests used for Presumptive Identification
    • Theory: Simple, quick assays detecting enzymatic activities or metabolic products. Examples: Catalase, Coagulase, Oxidase, Indole, PYR
    • Interpretation: Based on color changes, bubble formation, or other visual indicators (positive or negative results)
    • Application: Provides quick presumptive identification, guiding the selection of further tests and enabling faster initiation of appropriate therapy
    • Examples
      • Catalase: Differentiates Staphylococcus (positive) from Streptococcus (negative)
      • Coagulase: Differentiates Staphylococcus aureus (positive) from coagulase-negative staphylococci (CoNS)
      • Oxidase: Differentiates Neisseria and Moraxella (positive) from Enterobacteriaceae (negative)
      • Indole: Differentiates Escherichia coli (positive) from other Enterobacteriaceae
      • PYR: Differentiates Streptococcus pyogenes (positive) from other beta-hemolytic streptococci

Definitive Identification Methods

These methods provide more specific and reliable identification

  • Conventional Biochemical Identification
    • Theory: Assesses metabolic capabilities of bacteria using specific substrates. Examples: X and V factors, Neisseria carbohydrate utilization
    • Interpretation: Based on the presence or absence of enzymatic activity, substrate utilization (acid production, gas production), or growth requirements (e.g., X and V factors)
    • Application: Used after presumptive identification to confirm the identification, differentiate closely related species, and guide antibiotic selection
  • Commercial Kits
    • Theory: Prepackaged, standardized systems using various biochemical reactions in miniaturized formats
    • Interpretation: Reading results visually or with automated systems. Results are interpreted by matching the reaction pattern to a database
    • Application: Convenient, fast, and standardized for identifying a wide variety of organisms
    • Examples: API strips, Vitek, MicroScan
  • Automated Methods
    • Theory: Computer-controlled instruments performing multiple biochemical tests and/or advanced techniques
    • Interpretation: Instruments analyze data and provide an identification with a probability and/or confidence level
    • Application: High throughput, speed, efficiency, and objective results
    • Examples: Vitek 2, MicroScan, Phoenix
  • MALDI-TOF MS
    • Theory: Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry analyzes the protein profile of a bacterial cell, creating a unique “fingerprint.”
    • Interpretation: The mass spectrum is compared to a database of known bacterial spectra. Identification is based on a spectral match and a probability score
    • Application: Rapid, accurate, and cost-effective for routine identification of bacteria and yeast
  • Molecular Methods
    • Theory: Analyze the DNA or RNA of bacteria
    • Interpretation: Based on the presence or absence of target sequences, or the sequence itself
    • Application: High sensitivity, specificity, rapid results, culture-independent
    • Methods
      • PCR: Amplifies specific DNA sequences
      • qPCR: Real-time PCR with fluorescent detection, for quantitative analysis
      • Multiplex PCR: Amplifies multiple targets simultaneously
      • Sequencing (e.g., 16S rRNA gene sequencing): Determines the DNA sequence for identification and phylogenetic analysis
      • Whole Genome Sequencing (WGS): Determines the complete genome sequence for detailed strain typing, outbreak investigations, and identification of virulence factors and resistance genes
      • Nucleic Acid Hybridization: Detects specific DNA or RNA sequences using labeled probes
      • Microarrays: Detects multiple target sequences simultaneously
    • Sequencing (e.g., 16S)
      • Theory: Sequencing a highly conserved and variable region of the 16S rRNA gene
      • Interpretation: Percentage identity and a closest match are determined by sequence alignment to a database
      • Application: Species identification and phylogenetic analysis

Interrelationships and Integrated Approach

  • Sequential Process: The identification process is typically a sequential one. The Gram stain and colony morphology provide the initial clues. Rapid tests are then performed to provide presumptive identification. These results guide the selection of more definitive tests, such as conventional biochemical tests, commercial kits, automated methods, or molecular methods
  • Correlation: The results from all methods are correlated to reach a definitive identification. Discrepancies should be investigated
  • Choosing the Right Method: The choice of identification method depends on the clinical situation, the suspected organism, and the laboratory’s resources
  • Reporting: The final identification, along with any relevant susceptibility results, is reported to the clinician