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Microbiology

Preparation Methods

This section details specimen preparation methods and applications within preanalytic specimen processing, highlighting the key aspects of concentration, digestion/decontamination, cross-contamination prevention, sterile technique, tissue homogenization, and DNA/RNA extraction:

Core Concept

Proper specimen preparation is essential for accurate and reliable results in the clinical microbiology laboratory. It involves a series of steps designed to:

  • Isolate and concentrate target microorganisms or analytes
  • Remove interfering substances
  • Maintain the integrity of the sample
  • Prevent contamination

Concentration

  • Purpose: Increase the number of target organisms or analytes in a sample to improve the sensitivity of downstream tests
  • Methods
    • Centrifugation: Separates components based on density
    • Filtration: Traps microorganisms on a filter membrane
    • Sedimentation: Allows heavier particles to settle
    • Flotation: Uses a high-density solution to float target organisms
    • Immunomagnetic Separation: Uses antibodies to capture specific organisms
  • Applications
    • Urine for culture
    • CSF for Gram stain and culture
    • Stool for parasite detection
    • Water testing
  • Key Considerations: Sterility, recovery rate, and appropriate method selection based on specimen and target organism

Digestion/Decontamination

  • Purpose: Liquefy viscous specimens and selectively eliminate non-target organisms to facilitate the isolation of specific pathogens
  • Methods
    • NALC-NaOH: Liquefies mucus and decontaminates (mycobacteria)
    • Zephiran-Trisodium Phosphate: Similar to NALC-NaOH
    • Oxalic Acid: Inhibits non-mycobacterial growth
    • Sputolysin: Mucolytic agent
  • Applications
    • Sputum for Mycobacterium tuberculosis culture
    • Bronchial washings
  • Key Considerations: Reagent concentration, incubation time, and proper neutralization to avoid damaging target organisms

Prevention of Cross-Contamination

  • Purpose: Avoid the transfer of microorganisms from one sample or source to another, ensuring accurate results
  • Strategies
    • Aseptic technique: Minimize the introduction of contaminants
    • Unidirectional workflow: Separate clean and contaminated areas
    • PPE: Gloves, lab coats, eye protection
    • Hand hygiene: Frequent and thorough handwashing
    • Disinfection/Sterilization: Regular cleaning of surfaces and equipment
    • Proper waste disposal: Biohazard containers and sharps disposal
  • Key Considerations: Strict adherence to protocols, regular audits, and training

Sterile Technique

  • Purpose: Maintain a microbe-free environment during procedures to prevent contamination
  • Key Elements
    • Sterile Instruments and Equipment: Autoclaving, filtration, etc
    • Sterile Field: Creating and maintaining a sterile workspace
    • Hand Hygiene: Critical for preventing the spread of microorganisms
    • PPE: Gloves, masks, etc
    • Working in Biosafety Cabinets (BSCs): Provides a controlled environment
  • Applications
    • Culture inoculation
    • Subculturing
    • Reagent preparation
  • Key Considerations: Consistent adherence to protocols, proper training, and regular monitoring

Tissue Homogenization

  • Purpose: Disrupt the structure of tissue samples to release microorganisms, nucleic acids, proteins, and other analytes for downstream testing
  • Methods
    • Mechanical Homogenization: Grinding or using automated homogenizers
    • Enzymatic Digestion: Using enzymes to break down the extracellular matrix
    • Chemical Lysis: Using chemical agents to disrupt cell membranes
    • Ultrasonication: Using high-frequency sound waves
  • Applications
    • Microorganism recovery from biopsies
    • Nucleic acid extraction from tissue samples
  • Key Considerations: Tissue type, sample size, temperature control, and proper buffer selection

DNA/RNA Extraction

  • Purpose: Isolate and purify nucleic acids from a variety of biological samples for molecular testing
  • Methods
    • Organic Extraction (Phenol-Chloroform): Uses organic solvents
    • Solid-Phase Extraction (Silica-Based Columns): Uses a solid matrix to bind nucleic acids
    • Magnetic Bead-Based Extraction: Uses magnetic beads coated with a substance that binds nucleic acids
    • Automated Extraction Systems: Uses automated instruments
  • Applications
    • Pathogen detection
    • Genetic analysis
    • Diagnostic testing
    • Research
  • Key Considerations: Sample type, lysis method, binding efficiency, washing steps, elution volume, and quality control (quantification and purity)