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Microbiology

Microscopic ID

This is where the real detective work begins, as examining the microscopic structures of fungi is crucial for accurate identification. We’ll cover the key structures to look for, the staining techniques used to visualize them, and the characteristic features of major fungal pathogens

Key Microscopic Structures

When examining a fungal specimen under the microscope, focus on these structures:

  • Hyphae
    • Definition: The basic structural units of filamentous fungi. They are thread-like filaments that form the mycelium (the vegetative part of the fungus)
    • Septate vs. Non-Septate: Are the hyphae divided by cross-walls (septa)? Septate hyphae have distinct compartments, while non-septate hyphae are continuous tubes
    • Hyaline vs. Dematiaceous: Are the hyphae clear (hyaline) or dark-pigmented (dematiaceous)?
    • Branching: What is the pattern of branching? (e.g., dichotomous branching in Aspergillus)
  • Conidia
    • Definition: Asexual spores produced by fungi
    • Conidiophore: The specialized structure that bears the conidia. The shape and arrangement of conidiophores are important for identification
    • Types of Conidia
      • Microconidia: Small, single-celled conidia
      • Macroconidia: Large, multi-celled conidia
      • Arthroconidia: Conidia formed by the fragmentation of hyphae
      • Blastospores: Conidia formed by budding
    • Arrangement: How are the conidia arranged on the conidiophore? (e.g., chains, clusters, heads)
  • Yeast Cells
    • Definition: Single-celled fungi that reproduce by budding
    • Budding: The process of forming a new yeast cell from an outgrowth or bud on the parent cell
    • Pseudohyphae: Chains of elongated yeast cells that resemble hyphae but are formed by budding rather than true hyphal growth
    • Germ Tube: A short, hypha-like extension produced by Candida albicans when incubated in serum
  • Other Structures
    • Spherules: Large, round structures containing endospores, characteristic of Coccidioides immitis
    • Sclerotic Bodies: Copper-colored, round structures found in tissue samples from patients with chromoblastomycosis
    • Sporangia: Sac-like structures containing spores, produced by Mucorales
    • Rhizoids: Root-like structures that anchor the fungus to the substrate, characteristic of Rhizopus

Staining Techniques

Several staining techniques are used to visualize fungal structures:

  • KOH Preparation
    • Principle: Potassium hydroxide (KOH) dissolves keratin, making fungal elements more visible in skin, hair, and nail samples
    • Procedure: Place the specimen on a slide with a drop of 10-20% KOH. Gently heat the slide to speed up the clearing process
    • Visualization: Hyphae and conidia appear clear against a cleared background
  • Gram Stain
    • Principle: A general-purpose stain that differentiates bacteria based on cell wall structure. Fungi typically stain Gram-positive
    • Procedure: Follow standard Gram stain procedure
    • Visualization: Fungal hyphae and yeast cells stain purple
  • GMS (Gomori Methenamine Silver) Stain
    • Principle: Silver ions bind to the cell walls of fungi, creating a dark brown or black stain
    • Procedure: Tissue sections are stained with GMS reagent
    • Visualization: Fungal hyphae and spores stain black against a green background
  • PAS (Periodic Acid-Schiff) Stain
    • Principle: Periodic acid oxidizes carbohydrates in the fungal cell wall, which then react with Schiff reagent to produce a magenta color
    • Procedure: Tissue sections are stained with PAS reagent
    • Visualization: Fungal hyphae and spores stain magenta
  • India Ink Stain
    • Principle: A negative stain used to visualize the capsule surrounding Cryptococcus neoformans in CSF
    • Procedure: Mix a drop of CSF with a drop of India ink on a slide. Examine under a microscope
    • Visualization: The capsule appears as a clear halo around the yeast cell, as the ink particles cannot penetrate the capsule
  • Calcofluor White Stain
    • Principle: A fluorescent dye that binds to chitin in fungal cell walls
    • Procedure: Stain the specimen with calcofluor white and examine under a fluorescent microscope
    • Visualization: Fungal hyphae and spores appear bright white or blue against a dark background

Microscopic Features of Major Pathogens

Here’s a breakdown of the characteristic microscopic features of some common fungal pathogens:

Yeasts

  • Candida albicans
    • Yeast Cells: Round to oval yeast cells (3-6 μm)
    • Pseudohyphae: Chains of elongated yeast cells
    • Germ Tube: Produces a germ tube when incubated in serum
  • Cryptococcus neoformans
    • Yeast Cells: Round yeast cells (5-10 μm) with a thick capsule
    • Capsule: Visible with India ink stain
  • Malassezia furfur
    • Yeast Cells: Round to oval yeast cells (3-8 μm)
    • Hyphae: Short, stubby hyphae
    • “Spaghetti and Meatballs” Appearance: A mixture of yeast cells and short hyphae

Dimorphic Fungi

  • Histoplasma capsulatum
    • Mold Form (25-30°C)
      • Hyphae: Septate
      • Microconidia: Small, round to oval
      • Macroconidia: Tuberculate macroconidia (large, thick-walled conidia with knob-like projections)
    • Yeast Form (35-37°C)
      • Yeast Cells: Small, oval yeast cells (2-4 μm) found within macrophages
  • Blastomyces dermatitidis
    • Mold Form (25-30°C)
      • Hyphae: Septate
      • Conidia: Small, oval conidia on short conidiophores (“lollipop” appearance)
    • Yeast Form (35-37°C)
      • Yeast Cells: Large, round yeast cells (8-15 μm) with thick walls and broad-based budding
  • Coccidioides immitis
    • Mold Form (25-30°C)
      • Hyphae: Septate
      • Arthroconidia: Barrel-shaped arthroconidia that alternate with empty cells
    • Tissue Form (Spherules)
      • Spherules: Large, round structures (20-60 μm) containing numerous endospores
  • Sporothrix schenckii
    • Mold Form (25-30°C)
      • Hyphae: Septate, delicate
      • Conidia: Oval conidia arranged in a rosette-like pattern at the tips of conidiophores
    • Yeast Form (35-37°C)
      • Yeast Cells: Small, round or oval yeast cells (3-5 μm)

Dermatophytes

  • Trichophyton rubrum
    • Hyphae: Septate
    • Microconidia: Teardrop-shaped, along the sides of the hyphae
    • Macroconidia: Pencil-shaped, thin-walled, with 3-8 cells
  • Trichophyton mentagrophytes
    • Hyphae: Septate
    • Microconidia: Round to oval, in grape-like clusters
    • Macroconidia: Cigar-shaped, thin-walled, with 4-5 cells
  • Microsporum canis
    • Hyphae: Septate
    • Macroconidia: Spindle-shaped, thick-walled, with 8-15 cells, often with a pointed end

Mucorales (Zygomycetes)

  • Rhizopus spp.
    • Hyphae: Broad, ribbon-like, non-septate or sparsely septate
    • Sporangia: Round sporangia containing numerous spores
    • Rhizoids: Present, originating at the point where the stolon connects to the sporangiophore
  • Mucor spp.
    • Hyphae: Broad, ribbon-like, non-septate or sparsely septate
    • Sporangia: Round sporangia containing numerous spores
    • Rhizoids: Absent

Hyaline Molds

  • Aspergillus fumigatus
    • Hyphae: Septate, with dichotomous branching (branching at a 45-degree angle)
    • Conidiophore: Conidiophore with a flask-shaped vesicle and chains of conidia (phialides) covering the upper two-thirds of the vesicle
  • Fusarium spp.
    • Hyphae: Septate
    • Macroconidia: Sickle-shaped or canoe-shaped
  • Penicillium spp.
    • Hyphae: Septate
    • Conidiophore: Brush-like (penicillus) with chains of conidia
  • Scedosporium apiospermum
    • Hyphae: Septate
    • Conidia: Single-celled, oval (annelloconidia)

Dematiaceous Molds

  • Alternaria spp.
    • Hyphae: Septate, dark-pigmented
    • Conidia: Large, multi-celled, with both transverse and longitudinal septa. Often have a “drumstick” or “club-shaped” appearance
  • Cladosporium spp.
    • Hyphae: Septate, dark-pigmented
    • Conidia: One- or two-celled, forming branching chains
  • Fonsecaea spp.
    • Hyphae: Septate, dark-pigmented
    • Sclerotic Bodies: Copper-colored, round structures found in tissue samples
    • Conidiation: Exhibits multiple types of conidiation (Cladosporium-type, Phialophora-type, Rhinocladiella-type)

Pneumocystis jirovecii

  • Cysts: Round or oval cysts (5-8 μm) with a thick wall. Stain black with GMS stain
  • Trophozoites: Small, irregular-shaped cells (1-2 μm). Stain poorly with GMS stain, but can be visualized with Giemsa stain

Microsporidium spp.

  • Spores: Small, oval-shaped spores (1-4 μm). Stain pink to red with modified trichrome stain or bright white/blue with calcofluor white stain

Important Considerations

  • Experience: Microscopic identification requires experience and familiarity with the characteristic features of different fungal species
  • Reference Materials: Use reference materials (e.g., textbooks, atlases) to compare your observations with known characteristics
  • Multiple Structures: Examine multiple structures (hyphae, conidia, yeast cells) to confirm your identification
  • Correlation with Colony Morphology: Correlate the microscopic findings with the colony morphology to aid in identification
  • Molecular Methods: Molecular methods (PCR, sequencing) are increasingly used for definitive identification, especially for difficult-to-identify species

Key Takeaways

  • Microscopic identification is a crucial step in mycology
  • Focus on hyphae, conidia, yeast cells, and other characteristic structures
  • Use appropriate staining techniques to visualize fungal elements
  • Correlate microscopic findings with colony morphology
  • Use reference materials and experience to accurately identify fungal species

Key Terms

  • Hyphae: The thread-like filaments that form the mycelium of a fungus
  • Septate Hyphae: Hyphae that are divided into compartments by cross-walls (septa)
  • Non-Septate Hyphae: Hyphae that lack cross-walls (septa)
  • Conidia: Asexual spores produced by fungi
  • Conidiophore: A specialized hyphal structure that produces conidia
  • Yeast Cells: Single-celled fungi that reproduce by budding
  • Pseudohyphae: Chains of elongated yeast cells that resemble hyphae
  • Germ Tube: A short, hypha-like extension produced by Candida albicans
  • Spherules: Large, round structures containing endospores, characteristic of Coccidioides immitis
  • Sclerotic Bodies: Copper-colored, round structures found in tissue samples from patients with chromoblastomycosis
  • Sporangia: Sac-like structures containing spores, produced by Mucorales
  • Rhizoids: Root-like structures that anchor the fungus to the substrate, characteristic of Rhizopus
  • KOH Preparation: A microscopic technique where a sample is mixed with potassium hydroxide (KOH) to dissolve keratin
  • Gram Stain: A general-purpose stain that differentiates bacteria based on cell wall structure
  • GMS (Gomori Methenamine Silver) Stain: A staining technique used to visualize fungal cell walls
  • PAS (Periodic Acid-Schiff) Stain: A staining technique used to visualize carbohydrates in fungal cell walls
  • India Ink Stain: A negative stain used to visualize the capsule of Cryptococcus neoformans
  • Calcofluor White Stain: A fluorescent stain that binds to chitin in fungal cell walls
  • Dichotomous Branching: Branching that occurs at a 45-degree angle, characteristic of Aspergillus hyphae
  • Arthroconidia: A type of asexual spore formed by the fragmentation of hyphae, characteristic of Coccidioides
  • Maldi-TOF MS: Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
  • Panfungal PCR: is a molecular diagnostic technique that utilizes polymerase chain reaction (PCR) to amplify and detect conserved DNA sequences present in a wide range of fungi, allowing for the identification of fungal infections, even when traditional culture methods are negative or impractical