Micro IDs

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Chapter 6: Traditional Culture and Identification Methods

Culture Setup – Streak Plate Technique

General Procedure

  1. Gram Stain of Specimen

    • Report the following quantities:
      • Rare
      • Few
      • Moderate (Mod.)
      • Many
    • Certain specimens may require special preparation or specific Gram criteria, as discussed in Chapter 4.
    • STAT Considerations: Address any urgent implications as necessary.

  2. Plating Procedure

    • Plate the specimen on appropriate culture media, which varies by specimen type (see Chapter 4 for specifics).

  3. Incubation and Reading

    • Incubate under conditions suitable for the specimen type.
    • Read plates as per recommended guidelines specific to specimen type.

Streaking Methodology

  • Inoculation:
    • Use a single pass of a sterile swab near the edge of the plate.
  • Wire Loop Sterilization:
    • Heat the wire loop until it is red or glowing hot and allow it to cool fully.
    • It is crucial to repeat the sterilization technique between each pass or streaking area.
  • Creating Streaks:
    • With the sterilized loop, streak through the inoculated area in a zig-zag pattern, starting from the rim and moving toward the center, covering up to ¼ of the plate’s surface.
    • Press the loop gently against the agar, avoiding pitting or scratching the medium.
    • Second Streak: Pass the loop through the first area toward the rim and repeat the zig-zag motion.
    • Third Streak: Pass the loop through the second streaking area, again following the zig-zag pattern.
    • Fourth Streak: This final streak should be run through the third area, repeating the zig-zag pattern, but with a slightly wider spacing between each line created in this pass.
  • This technique results in isolated colony growth to facilitate proper identification.

Culture – Streak Plate Technique

  • The process described in the general procedure is repeated for clarity.
  • Emphasis on inoculation, wire loop sterilization, and the detailed streaking process ensures proper colony isolation for identification purposes.

Streak Plate Area Designation

AreaResulting Growth
Area 1Heavy Confluent Growth
Area 2Less Dense Growth
Area 3Weak Growth
Area 4Isolated Single Colonies

Phenotypic Characteristics

  • Microscopic Morphology:
    • Evaluation of microscopic morphology and staining characteristics, particularly the Gram stain reaction.
  • Colonial Morphology:
    • Examination of appearance on artificial plating media.
  • Environmental Requirements:
    • Understanding the growth requirements such as oxygen levels (e.g. capnophilic organisms).
  • Nutritional Needs:
    • Whether the organisms require enriched or supplemented media.
  • Antimicrobial Susceptibility:
    • Information on whether the organisms are resistant or sensitive to specific antibiotics.

Colonial Morphology Features

  • Key characteristics to observe include:
    • Hemolysis
    • Size
    • Form or Margin
    • Elevation
    • Surface
    • Consistency
    • Density
    • Color

Pigmentation of Specific Organisms

  • Pseudomonas aeruginosa: Produces a blue-green pigment.
  • Serratia marcescens: Exhibits red pigmentation.
  • Chromobacter violaceum: Results in purple pigmentation.
  • Prevotella melaninogenica: Characterized by a brown to black pigmentation.

Colonial Characteristics Examples

  1. Proteus swarming on sheep blood agar, known for its characteristic swarming pattern.
  2. Klebsiella pneumoniae exhibits mucoid morphology, visible on both sheep blood agar and MacConkey agar.

Preliminary Biochemical Tests

  • Carbohydrate Utilization: Evaluating specific enzyme functionalities.

    • Enzymes tested:
      • Fermentation
      • Oxidation
    • Indicators Used:
      • pH indicators
      • Gas production
    • Triple Sugar Iron (TSI) Media:
      • Example:
      • Tube on the left is not inoculated.
      • Tube on the right shows acid/acid with gas production indicating fermentation of glucose and lactose/sucrose with gas.

  • Catalase Test:

    • Converts hydrogen peroxide (H2O2) to water and oxygen (bubbles)
    • Important for differentiating:
      • Staphylococcus (+) from Streptococcus (-)
      • Bacillus (+) from Clostridium (-)
    • Positive reaction observed as bubbles in response to hydrogen peroxide exposure.

  • Coagulase Test:

    • Converts fibrinogen to fibrin.
    • Types:
      • Slide test (Bound coagulase)
      • Tube test (Free coagulase)
    • Differentiates Staphylococcus aureus from coagulase-negative staphylococci.

  • Cytochrome Oxidase Test:

    • The enzyme oxidizes tetramethyl-para-phenylenediamine dihydrochloride in the presence of oxygen to form indophenol.
    • Organisms that are oxidase positive include Neisseria, Pseudomonas, Campylobacter, Aeromonas, and Pasteurella.
    • Enterobacteriaceae are typically oxidase negative.

  • Spot Indole Test:

    • Detects the enzyme tryptophanase which degrades tryptophan on blood agar or chocolate agar.
    • Procedure: Smear colony on filter paper, apply Kovach’s reagent; a positive result is indicated by a blue to violet color.
    • Important for differentiating:
      • E. coli (+) from other lactose-fermenting Enterobacteriaceae.
      • Proteus mirabilis (-) from Proteus vulgaris (+).

  • PYR Hydrolysis Test:

    • L-pyrrolidonyl-B-naphthylamide (PYR) is broken down by L-pyrroglutamyl aminopeptidase.
    • Differentiates between:
      • Group D streptococcus (Enterococcus (+) vs. non-Enterococcus (-)).
      • Beta hemolytic streptococcus (Group A (+) vs. Group B (-)).
    • Positive reaction is indicated by a pink color.

Multitest Systems

Features:

  • Encompass manual and semi-automated procedures for the inoculation, incubation, interpretation, and results reporting.
  • Offer shorter incubation times compared to tubed media.
  • Utilize biochemical and enzymatic reactions for identification.
  • Provide more reproducible and consistent results (i.e., one day versus three days).

Examples:

  • bioMérieux API®: Microtubes with dehydrated substrates reconstituted with bacterial suspension; systems designed for a variety of bacteria including Enterobacteriaceae, non-fermenters, Neisseria, Haemophilus, Staphylococcus, Streptococcus, Corynebacteria, anaerobes, and yeast.
  • Access databases via the Internet-based APIweb service for identification and reporting.

Detection of Metabolic Activity

  • Colorimetry:
    • Spectrophotometers measure color changes indicative of pH indicator reactions or chromogenic substrates.
  • Nephelometry:
    • Light scattering measurement useful for detecting turbidity, crucial in antibiotic susceptibility testing.
  • Fluorometry:
    • Substrates bound to a fluorophore fluoresce as metabolic processes occur, represents bacterial activity.

Automated Systems

Overview:

  • Systems designed for identification and antibiotic susceptibility testing; vary in complexity based on desired testing volume.
  • Utilize principles of colorimetry, nephelometry, and fluorometry for efficient analysis.

Specific Systems:

  • MicroScan® System (Siemens Medical Solutions Diagnostics)
  • Sensititre System (TREK Diagnostic Systems)
  • BD Phoenix Automated Microbiology System
  • VITEK® System (bioMérieux VITEK)
  • MicroScan WalkAway® (Siemens Medical Solutions Diagnostics)

MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry)

Principle:

  1. Specimen applied to target plate.
  2. Proteins and cellular components extracted for mass analysis.
  3. Target plate passes through the ionization chamber where laser ionizes samples in pulses.
  4. Proteins released as singly charged ions.
  5. These ions pass through a vacuum tube and contact a detector; the time taken for ions to travel through the tube is referred to as "time of flight".
  6. A spectrum generated based on the mass-to-charge ratio of the molecules.
  7. The spectrum is species-specific, digitized, and compared to a database for identification.

Figure Reference:

  • Figure 6-14 shows the BBL Crystal System (BD BBL Diagnostics).

Manual Blood Cultures

  • Manual systems tailored for smaller volume laboratories include:
    • Septi-Chek Blood Culture System by Becton Dickinson
    • Oxoid SIGNAL Blood Culture System by Thermo-Fisher Scientific
    • Isolator: Utilizes a lysis centrifugation method by Wampole Medical Professional Diagnostics.

Automated Blood Culture Systems

Examples and Technologies:

  1. BACTEC by Becton Dickinson:

    • The first automated blood culture system. Early models (e.g., BACTEC 460) employed a radiometric principle based on 14CO2 release from 14C radioactively labeled substrates.
    • Advanced versions (e.g., BACTEC 660, NR 860) directly detect CO2 release using infrared spectrophotometry.
    • The newest versions (e.g., BACTEC 9240, 9120) fully automate the measurement of released CO2 or consumed oxygen using fluorescence.

  2. BacT/ALERT® 3D and VIRTUO® by bioMérieux:

    • Utilize colorimetric technology to photometrically measure microbial-produced CO2.

  3. VersaTREK® System by Trek Diagnostic Systems:

    • Monitors pressure changes in the bottle headspace caused by bacterial metabolism.