Bacterial identification methods

Bacterial identification methods

Bacterial identification methods

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Stains & Cultures - Seeing is Believing

  • Gram Stain: Differentiates bacteria based on cell wall peptidoglycan content.

Gram stain: Gram-positive cocci and Gram-negative rods

  • Other Key Stains:
    • Ziehl-Neelsen: Acid-fast stain for Mycobacterium (pink/red on blue background).
    • India Ink: Negative stain for cryptococcal capsules.
    • Giemsa: For intracellular organisms. 📌 Giemsa helps you Get inside the cell (Chlamydia, Rickettsia).

⭐ Certain bacteria, like Legionella pneumophila, stain poorly with Gram stain and require specialized silver stains or growth on specific media like Buffered Charcoal Yeast Extract (BCYE) agar.

Biochemical Tests - What Bacteria Eat

  • Catalase: Differentiates Staphylococcus (CAT-positive) from Streptococcus (CAT-negative).
    • Degrades $H_2O_2$ into $H_2O$ and $O_2$ (bubbles).
  • Coagulase: Differentiates S. aureus (coagulase-positive) from other staphylococci.
  • Urease: Identifies urease-producers like Proteus, Klebsiella, and H. pylori.
    • 📌 Pee CHUNKSS: Proteus, Cryptococcus, H. pylori, Ureaplasma, Nocardia, Klebsiella, S. epidermidis, S. saprophyticus.
  • Oxidase: Identifies organisms with cytochrome c oxidase, e.g., Pseudomonas, Neisseria.
  • Indole: Differentiates E. coli (positive) from other lactose-fermenting enterics.

⭐ The CAMP test identifies Streptococcus agalactiae (Group B Strep). It shows an arrowhead-shaped zone of enhanced hemolysis when grown near a streak of S. aureus.

CAMP test showing positive and negative results

Immunologic Methods - Antibody Detectives

Utilizes the high specificity of antibody-antigen binding for identification. Can detect either bacterial antigens or host antibodies.

  • ELISA (Enzyme-Linked Immunosorbent Assay):

    • Highly sensitive method to detect antigens or antibodies.
    • Commonly used for HIV, Lyme disease, and viral hepatitis serology.
    • 📌 Sandwich ELISA: Traps the antigen between two antibodies.
  • Agglutination:

    • Visible clumping of antibody-coated particles (latex) or bacteria.
    • Used in rapid strep tests and for serotyping (e.g., Lancefield grouping, Salmonella serovars).
  • Immunofluorescence (IF):

    • Antibodies labeled with fluorescent dyes visualize bacteria under a microscope.
    • Direct (DFA) or Indirect (IFA) methods.

⭐ Serotyping is critical for epidemiology. E. coli O157:H7 is a key example; the O-antigen is from the lipopolysaccharide (LPS) and the H-antigen is from the flagella.

Direct vs. Indirect Immunofluorescence Microscopy

Molecular Methods - DNA Fingerprinting

  • Principle: Exploits variations in DNA sequences to create a unique genetic "fingerprint" for each bacterial strain, essential for epidemiological tracking.
  • Core Techniques:
    • Restriction Fragment Length Polymorphism (RFLP):
      • Bacterial DNA is digested by restriction enzymes.
      • Resulting fragments are separated by gel electrophoresis, creating a specific banding pattern.
    • Pulsed-Field Gel Electrophoresis (PFGE):
      • A specialized RFLP for separating very large DNA fragments using a periodically changing electric field.
    • Ribotyping:
      • An RFLP method targeting the highly conserved ribosomal RNA (rRNA) genes.

DNA Fingerprinting Gel Electrophoresis of Bacterial Strains

High-Yield: PFGE is considered the gold standard for molecular subtyping in many epidemiological investigations, crucial for tracking outbreaks of pathogens like Listeria monocytogenes, Salmonella, and E. coli O157:H7.

High‑Yield Points - ⚡ Biggest Takeaways

  • Gram stain is the crucial first step, dividing bacteria into Gram-positive (purple) and Gram-negative (pink) based on peptidoglycan wall structure.
  • Catalase test differentiates Staphylococcus (+) from Streptococcus (-).
  • Coagulase test separates S. aureus (+) from coagulase-negative staphylococci.
  • Lactose fermentation on MacConkey agar distinguishes fermenters like E. coli from non-fermenters like Salmonella.
  • Hemolysis patterns (α, β, γ) are key for classifying Streptococcus species.
  • Acid-fast stain is essential to identify Mycobacterium species.

Practice Questions: Bacterial identification methods

Test your understanding with these related questions

A 28-year-old man comes to the physician for a pre-employment examination. He has no history of serious illness and takes no medications. A screening blood test is performed in which peptides are added to the sample to stimulate in vitro production of interferon-gamma, which is then measured using an enzyme-linked immunosorbent assay. This test is most likely to be helpful in diagnosing infection with which of the following pathogens?

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Flashcards: Bacterial identification methods

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The Weil-Felix test tests the cross reactivity of rickettsia and _____ OX19 and OX2

TAP TO REVEAL ANSWER

The Weil-Felix test tests the cross reactivity of rickettsia and _____ OX19 and OX2

proteus vulgaris

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