Pathogenic Microbiology: intro to molds/yeasts

Introduction to Mycology and Laboratory Diagnosis of Fungal Infection

Background of Fungi

  • Characteristics of Fungi:

    • Eukaryotic organisms that possess the following traits:

    • Absorb nutrients from their environment.

    • Cell walls contain chitin

    • Prefer neutral pH for growth

    • Require moist environments for growth; however, conidia (asexual fungal reproductive structures) can survive in dry environments.

    • Classified as saprobes or saprophytes, meaning they primarily consume dead organic matter

Yeasts

  • Description:

    • Yeasts are single cells that reproduce by budding.

  • Growth characteristics:

    • They form smooth, creamy, and bacterial-like colonies without aerial hyphae.

  • Reproductive methods:

    • Budding: In this process, a new daughter cell (known as a blastoconidium) is formed as a bud matures.

    • Fission: Involves the splitting of a cell into two equal-sized cells, which then grow from the tips of each.

Molds

  • Appearance:

    • Characterized by a fuzzy or wooly appearance due to the collection of intertwined hyphae forming a mycelium.

  • Hyphae structure:

    • Aerial hyphae: These hyphae extend above the surface of the colony and support reproductive structures that produce conidia.

    • Vegetative hyphae: These hyphae extend downward into the growth medium to absorb nutrients.

  • Microscopic appearance:

    • Different shapes of hyphae are crucial for identification:

    • Antler: Swollen, branching tips.

    • Racquet: Enlarged, club-like areas.

    • Rhizoids: Root-like structures commonly found in Zygomycetes.

    • Spiral: Tightly coiled structure.

  • Septation of Hyphae:

    • Septate Hyphae: Hyphae with frequent cross-walls, or septa, occurring perpendicularly to their outer walls.

    • Aseptate Hyphae: These hyphae have no septations, separating them from septate hyphae.

    • Sparsely septate: Few cross-walls at irregular intervals.

Pigmentation

  • Types of Hyphae:

    • Hyaline (moniliaceous): Non-pigmented or lightly pigmented hyphae.

    • Phaeoid (dematiaceous): Darkly pigmented hyphae (contains melanin), ranging from pale brown to black.

  • Fontana-Masson stain:

    • This stain is used to stain melanin; phaeoid molds appear darker brown, and hyaline hyphae appear in pink/red

Dimorphism and Polymorphism

  • Dimorphism:

    • Refers to the existence in two forms:

    • Mold phase: Grows at temperatures of 22-25°C.

    • Yeast phase: Grows at 37°C, the optimal temperature for tissue states in vivo.

  • Polymorphism:

    • Refers to different forms of fungi (yeast and mold) present in the same culture,

Fungal Reproduction

  • Asexual reproduction:

    • Known as imperfect: formation of conidia following mitosis.

    • Fruiting structures:

    • Conidiogenous cells:

      • Phialides: Vase-like structures producing phialoconidia.

      • Annellides: Ringed structures producing annelloconidia.

    • Arthroconidia: Result from the fragmentation of fertile hyphae.

  • Sexual reproduction (Fungi Perfecti):

    • Fungi can reproduce both sexually and asexually.

Direct Microscopic Examination

  • Common methods:

    • KOH prep:

    • KOH solution (10-20%): Breaks down keratin in skin, hair, or nails, liberating fungal elements for easier examination.

    • Calcofluor white: Often combined with KOH for fluorescent microscopy; can detect chitin in fungal cell walls.

    • India Ink: Primarily used to examine CSF for encapsulated yeasts like C. neoformans.

  • Tissue Stains:

    • Periodic Acid Schiff (PAS): Stains polysaccharides in the fungal cell wall pink.

Fungal Culture Media

  • Common media used:

    • Sabouraud dextrose agar (SDA): Neutral pH, efficient for primary fungal isolation, may contain antimicrobial agents to suppress bacterial growth.

  • Handling and Incubation:

    • Incubate at room temperature to 30°C for optimal fungi growth, but 37°C is used for specialized temperature studies.

Evaluation Techniques

  • Direct mounts:

    • Tease mount: Using needles to separate mycelium in lactophenol cotton blue.

    • Cellophane tape preparation: press tape onto the colony→ place onto drop of lacophenol cotton blue

Antifungal Drugs

  • Types of antifungal medications:

    • Polyenes: amphotericin B

    • Azoles

    • Echinocandins: caspofungin

    • Allylamines: terbinafine and naftifine.