Fungus Identification in the Laboratory
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Direct answer
KOH dissolves keratin and leaves fungus standing: a 10-20 per cent potassium hydroxide mount (20-40 per cent with DMSO for nails) of skin scraping, hair or nail clears the specimen within minutes to an hour and shows hyphae, spores or budding yeast directly — the fastest dermatophyte answer there is. Lactophenol cotton blue (LPCB) is the identification mount for cultured fungi, staining septate hyphae and characteristic conidia; Sabouraud dextrose agar (with chloramphenicol to suppress bacteria and cycloheximide to suppress saprophytic moulds) is the culture medium, incubated at 25-30 degrees for moulds and 37 degrees for yeasts and dimorphic phase conversion. Yeast speciation runs through the germ tube test — germ tubes within two to three hours at 37 degrees in serum identify Candida albicans — CHROMagar colour differentiation, the tube-to-hyphae cornmeal morphology, and auxanographic sugar assimilation. For Cryptococcus, India ink or nigrosin on CSF shows the encapsulated budding yeast, latex agglutination for cryptococcal antigen is more sensitive, and the Indian context adds Talaromyces (Penicillium) marneffei in immunosuppressed patients from the north-east.
What you must remember
- Direct mounts by specimen: KOH with DMSO for skin, hair and nail (hair invasion types — endothrix versus ectothrix — read directly); calcofluor white fluorescence where available; India ink or nigrosin for encapsulated Cryptococcus in CSF.
- Culture setup: Sabouraud dextrose agar, chloramphenicol (antibacterial) plus cycloheximide (anti-saprophytic mould) combinations; incubate moulds at 25-30 degrees Celsius, dimorphic fungi in parallel at both temperatures, and speciate budding yeast at 37 degrees.
- Germ tube test: yeast in serum at 37 degrees Celsius for 2-3 hours; a germ tube (a hypha-like outgrowth without a constriction at the neck and without a septum) identifies Candida albicans, with rare false positives from Candida dubliniensis and occasional C. tropicalis outgrowths.
- CHROMagar Candida: species-specific colony colours — C. albicans green, C. tropicalis blue with pink halo, C. krusei pink, rough-edged — a presumptive speciation plate.
- LPCB morphology anchors: Microsporum macroconidia thick-walled rough; Trichophyton microconidia numerous; Epidermophyton smooth thin-walled macroconidia in clusters with no microconidia — the three dermatophyte genera separated at a stroke.
- Dimorphic fungi: Sporothrix, Histoplasma, Blastomyces and Talaromyces marneffei convert mould-to-yeast with temperature (25 to 37 degrees) and stain as intracellular yeasts in tissue; India reports sporotrichosis and histoplasmosis pockets and T. marneffei in HIV.
- Yeast versus mould first question: germ tube, then CHROMagar, then carbohydrate assimilation for the final call — with Candida auris colonisation and resistance an emerging hospital preoccupation.
Where marks leak
The germ tube is over-called: the mark goes to the definition — no basal constriction, no septum; chains of blastoconidia are not germ tubes. The second leak is KOH patience: a nail mount read at five minutes misses the fungus that appears after an hour of clearing. Third, the cycloheximide logic inverted: the reason contaminated specimens go onto cycloheximide-containing media is to suppress the fast saprophytic moulds that would otherwise overrun slow pathogens — asked as a mechanism, not a menu. And the emerging-answer bonus examiners reward: naming Candida auris as the multidrug-resistant, outbreak-associated yeast that colonises hospital surfaces and demands identification and notification — a modern Indian hospital preoccupation worth one prepared sentence in any viva.
Frequently asked questions
What is the germ tube test and which yeast does it identify?
Yeast incubated in serum at 37 degrees Celsius for 2-3 hours; a germ tube without basal constriction or septum identifies Candida albicans directly.
Why is DMSO added to KOH for nail specimens?
Keratin-dense nails clear too slowly in plain KOH; dimethyl sulphoxide accelerates penetration, dissolving keratin so hyphae become visible within minutes rather than an hour.
What media and additives are used for fungal culture?
Sabouraud dextrose agar with chloramphenicol to suppress bacteria and cycloheximide to suppress saprophytic moulds, incubated at 25-30 degrees Celsius for moulds and 37 degrees for yeasts.
How are the three dermatophyte genera separated microscopically?
In LPCB mounts: Microsporum by rough thick-walled macroconidia, Trichophyton by many microconidia, and Epidermophyton by smooth thin-walled macroconidia in clusters with no microconidia.
How is cryptococcal meningitis confirmed in the laboratory?
India ink or nigrosin negative staining showing encapsulated budding yeast in CSF, with the more sensitive cryptococcal antigen test (latex agglutination or lateral flow) on CSF or serum.