Mycology Lab Quiz
Fungal morphologic features, KOH mounts, Sabouraud agar, mold identification, and susceptibility testing.
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Mycology Lab Quiz: Fungal Morphology, KOH Mounts, Sabouraud Agar, Mold ID, and Susceptibility Testing
Fungal infections were, for much of medical history, considered relatively rare and limited mostly to superficial skin and nail infections in healthy people. That picture has changed dramatically. The global population of people living with immunosuppression due to HIV, organ transplantation, cancer chemotherapy, and increasingly, intensive care unit stays has grown substantially. For these patients, opportunistic fungi that would cause no illness in a healthy person can cause life-threatening invasive disease. The global burden of serious fungal infections now exceeds 300 million cases per year, with over 1.5 million deaths annually according to estimates by the Global Action Fund for Fungal Infections (GAFFI).
This quiz is designed for medical mycology students, clinical laboratory scientists, and infectious disease and dermatology learners who want to strengthen their understanding of the laboratory methods used to detect and identify fungi. The questions cover fungal morphology and the classification of fungi into yeasts, moulds, and dimorphic fungi, the KOH preparation and India ink stain, Sabouraud dextrose agar and its uses, identification of clinically important moulds by their microscopic features, and antifungal susceptibility testing methods.
Core Topics
Fungal Morphology: Yeasts, Moulds, and Dimorphic Fungi
Fungi exist in two main growth forms. Yeasts are single-celled fungi that reproduce by budding: the parent cell forms an outgrowth (a bud) that enlarges and eventually separates to form a daughter cell. On Gram stain, yeasts appear as oval to round cells, often with visible buds. The most clinically important yeast is Candida albicans. On Sabouraud agar, it produces smooth, cream-coloured colonies. In serum at 37 degrees Celsius, C. albicans produces a germ tube, a short hyphal extension, within 3 hours, which is a rapid and specific identification test.
Moulds grow as multicellular filamentous structures called hyphae, which interweave to form a visible mass called a mycelium. Hyphae may be septate (divided by cross-walls at regular intervals) or aseptate (without cross-walls, also called coenocytic). This distinction is clinically important: Aspergillus species and dermatophytes have septate hyphae, while Mucor and Rhizopus (agents of mucormycosis) have aseptate or sparsely septate hyphae. The spore-bearing structures, called conidia, vary in shape, arrangement, and size between different mould species and are essential for species identification.
Dimorphic fungi exist as yeasts at body temperature (37 degrees Celsius) and as moulds in the environment (25 degrees Celsius). This temperature-dependent morphological switch is a key virulence feature. Clinically important dimorphic fungi include Histoplasma capsulatum (causing histoplasmosis, primarily a lung infection endemic in parts of the Americas), Coccidioides immitis (causing coccidioidomycosis, endemic in the southwestern USA and parts of Latin America), Blastomyces dermatitidis (blastomycosis), and Talaromyces marneffei (an important opportunistic pathogen in Southeast Asia, formerly called Penicillium marneffei).
KOH Preparation and India Ink Stain
The potassium hydroxide (KOH) preparation is a rapid direct microscopy technique used to detect fungal elements in clinical specimens including skin scrapings, hair, nail clippings, sputum, and other material. KOH dissolves the keratin in the specimen, clearing the background and making fungal hyphae or yeast cells more visible. Calcofluor white, a fluorescent brightener, is often added to the KOH preparation to further enhance visualisation of fungal cell walls under fluorescence microscopy. A positive KOH preparation showing branching septate hyphae in a skin scraping from a ring-shaped scaly rash is sufficient to diagnose dermatophytosis (ringworm) without waiting for culture results.
The India ink stain is used specifically for detecting Cryptococcus neoformans in cerebrospinal fluid (CSF). Cryptococcus has a large polysaccharide capsule that excludes the India ink particles, producing a clear halo around the yeast cell visible against the dark background. This is one of the few situations in clinical microbiology where a direct stain can provide an immediate, specific diagnosis of a serious infection. Cryptococcal meningitis is a leading cause of death in people living with HIV in sub-Saharan Africa and Southeast Asia.
Sabouraud Dextrose Agar
Sabouraud dextrose agar (SDA) is the standard culture medium for fungi. It has a low pH (around 5.6) and a high glucose concentration that supports fungal growth while inhibiting many bacteria. Cycloheximide is sometimes added to suppress environmental saprophytic fungi, allowing isolation of dermatophytes from specimens contaminated with environmental moulds. Chloramphenicol is often added to suppress bacterial growth. SDA is incubated at both 25 to 30 degrees Celsius (to grow environmental and dimorphic mould forms) and 35 to 37 degrees Celsius (to grow yeasts and the yeast phase of dimorphic fungi). Fungal cultures typically take longer than bacterial cultures: yeasts may be visible in 2 to 5 days, while some moulds and dimorphic fungi may require 2 to 4 weeks.
Antifungal Susceptibility Testing
Antifungal susceptibility testing determines the minimum inhibitory concentration (MIC) of antifungal agents against a fungal isolate. The major antifungal classes include azoles (fluconazole, voriconazole, itraconazole, posaconazole), which inhibit ergosterol synthesis; echinocandins (caspofungin, micafungin, anidulafungin), which inhibit synthesis of the fungal cell wall component beta-glucan; polyenes (amphotericin B), which bind ergosterol and disrupt the fungal cell membrane; and flucytosine, which inhibits fungal DNA synthesis. Clinical breakpoints for interpretation are defined by EUCAST and CLSI.
Frequently Asked Questions
What is the difference between a yeast and a mould?
A yeast is a single-celled fungus that reproduces primarily by budding. A mould is a multicellular filamentous fungus that grows by extending branching hyphae to form a mycelium. Both types cause human infections. Yeasts cause infections like candidiasis and cryptococcosis. Moulds cause infections including aspergillosis, mucormycosis, and dermatophytosis. Some fungal species are dimorphic, existing as yeasts at body temperature and moulds at lower environmental temperatures.
What is a dimorphic fungus?
A dimorphic fungus can switch between two growth forms depending on temperature: it grows as a mould in the environment (at around 25 degrees Celsius) and as a yeast or yeast-like form at body temperature (37 degrees Celsius). This thermal dimorphism is an important virulence characteristic. Clinically important dimorphic fungi include Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, and Talaromyces marneffei.
What does a KOH preparation show?
A KOH preparation dissolves the background material of a clinical specimen (particularly keratin in skin, hair, and nails) while leaving fungal structures intact. Under microscopy, it reveals fungal hyphae, yeast cells, or spores. A positive KOH showing branching septate hyphae in a skin scraping supports a diagnosis of dermatophytosis. Adding calcofluor white causes fungal cell walls to fluoresce under UV light, improving sensitivity.
What is Sabouraud agar used for?
Sabouraud dextrose agar (SDA) is the primary isolation medium for fungi. Its acidic pH and high glucose content support fungal growth while inhibiting most bacteria. It is used to culture fungi from clinical specimens including skin and nail scrapings, vaginal swabs, sputum, urine, and blood cultures. Different fungal species produce characteristic colony morphology, colour, texture, and growth rate on SDA that contribute to their identification.
What is Aspergillus fumigatus and why is it dangerous?
Aspergillus fumigatus is a mould that produces enormous numbers of small airborne conidia (spores) that are constantly inhaled by everyone. In healthy individuals, the conidia are cleared by the immune system. In immunocompromised patients, particularly those with prolonged neutropenia or high-dose corticosteroid therapy, the conidia germinate and invade lung tissue, causing invasive pulmonary aspergillosis, which has a mortality rate of 30 to 90 per cent depending on the severity of immunosuppression. Azole-resistant A. fumigatus is an emerging and serious problem.
What is Candida and how is it identified in the lab?
Candida is a genus of yeasts that normally lives commensally on the skin, mucous membranes, and in the gastrointestinal tract. Candida albicans is the most common pathogenic species. In the lab, Candida species appear as Gram-positive oval budding yeast cells, sometimes with pseudohyphae (elongated yeast cells that resemble hyphae). C. albicans is identified by germ tube production in serum at 37 degrees Celsius, growth and morphology on chromogenic candida agar, and increasingly by MALDI-TOF or molecular testing.
What is an india ink stain used for?
The India ink stain is used to detect Cryptococcus neoformans in cerebrospinal fluid (CSF). The cryptococcal polysaccharide capsule repels the ink particles, producing a clear halo around the yeast cell visible against the dark ink background. A positive India ink preparation in CSF from a patient with meningitis symptoms provides a rapid presumptive diagnosis of cryptococcal meningitis. It is confirmed by cryptococcal antigen testing (CrAg) in CSF or serum, which is more sensitive.
What is dermatophytosis?
Dermatophytosis is a superficial fungal infection of the skin, hair, or nails caused by a group of fungi called dermatophytes (Trichophyton, Microsporum, and Epidermophyton species). Colloquially known as ringworm (tinea corporis), athlete’s foot (tinea pedis), jock itch (tinea cruris), or nail fungus (tinea unguium or onychomycosis), these infections are among the most common fungal infections worldwide. Dermatophytes digest keratin, the protein that makes up skin, hair, and nails, allowing them to thrive in these tissues.
How is Cryptococcus neoformans identified?
In the lab, Cryptococcus is identified by its encapsulated yeast morphology on India ink stain, its distinctive mucoid colony appearance on SDA (due to the large polysaccharide capsule), and growth on Niger seed agar (birdseed agar) where it produces brown pigmented colonies due to phenol oxidase activity. Cryptococcal antigen testing in CSF and serum using the lateral flow assay (CrAg LFA) is fast, highly sensitive, and specific. MALDI-TOF can identify Cryptococcus from pure culture.
What is a conidium?
A conidium (plural: conidia) is an asexual reproductive spore produced by moulds. Conidia are shed from specialised spore-bearing structures and can be dispersed through the air. The morphology of conidia, including their size, shape, arrangement, and the structures they are borne on, is the key feature used to identify moulds in the clinical laboratory. For example, Aspergillus fumigatus produces small spherical conidia in chains on flask-shaped structures called phialides arranged on a vesicle. Fusarium produces distinctive curved (sickle-shaped) macroconidia.