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Mycology – Yeast Student Lab Division of Medical Technology Carol Larson MSEd, MT(ASCP) Basic Characteristics • Unicellular • Generally normal flora Basic Characteristics • Asexual reproduction – Blastoconidia – Pseudohyphae – Arthroconidia Direct Examination • Observe for reproductive structures • Gram stain – Gram positive • India ink stain – Capsules Growth Requirements • Growth in 2-3 days • Temperature – 25-37ºC – Best at 30ºC Colony Morphology • Appearance – Similar to bacteria colony • Texture – Glabrous – May be filamentous • Pigmentation • Confuse with Staphylococcus Yeast ID Methods • Germ Tube Production – First test performed when yeast – Procedure – Interpretation Yeast ID Methods • Cornmeal-Tween 80 agar – Procedure – Observe microscopically for • • • • Chlamydospores Blastoconidia Pseudohyphae Arthrospores Yeast ID Methods • Niger Seed Agar (Birdseed agar) – Procedure – Observe for brown pigment caused by phenol oxidase activity – Cryptococcus neoformans Yeast ID Methods • Urease – Procedure – Positive: • Cryptococcus sp. (rapid) • Trichosporon sp. • Rhodotorula sp. – Negative: • Candida sp. • Geotrichum sp. Yeast ID Methods • Carbohydrate assimilation – Utilize carbohydrate as sole source of carbon in the presence of oxygen – Positive = growth • Carbohydrate fermentation – Utilize a carbohydrate anaerobically – Positive = gas Yeast ID Methods • Commercial identification kits – Manual methods – Automated methods Candida species • Normal flora • Leading opportunistic fungal infection in man • Candidiasis – Mucocutaneous – Cutaneous – Systemic Factors predisposing to infection • • • • • • • Immunocompromised Malignancy Prolonged antibiotic therapy Lupus, Diabetes Pregnancy and use of birth control pills Age Damaged skin barrier Candida albicans • #1 fungal isolate in laboratory • Germ tube: positive • CMT: clustered blastoconidia at septa of pseudohyphae, terminal chlamydospores • CHO: sucrose positive Candida albicans Candida stellatoidea • Germ tube: positive • CMT: clustered blastoconidia at septa of pseudohyphae, terminal chlamydospores • CHO: sucrose negative Candida tropicalis • Germ tube: negative • CMT: sparse single or short-chained blastoconidia anywhere along pseudohyphae, rare chlamydospores • CHO: sucrose positive Candida glabrata • Causes UTI • Germ tube: negative • CMT: only blastoconidia, no pseudohyphae • CHO: only glucose and trehalose positive Candida krusei • Germ tube: negative • CMT: tree-like branching of abundant blastoconidia from the septa of elongated pseudohyphae (“crossmatchsticks” appearance) Candida pseudotropicalis • Germ tube: negative • CMT: branched pseudohyphae with chains of elongated blastoconidia – logs in a stream arrangement of broken up blastoconidia positioned parallel to each other Candida parapsilosis • Germ tube: negative • CMT: few single or small clustered blastoconidia at or between septa of thin curved pseudohyphae. Sometimes giant pseudohyphae may be observed Cryptococcus neoformans • Cryptococcosis – Pulmonary – Meningitis • Pigeon droppings Cryptococcus neoformans • Gram stain • India ink stain Cryptococcus neoformans • Germ tube: negative • CMT: large, round blastoconidia, no pseudohyphae • Urease: positive (within 3 hours) Cryptococcus neoformans • Niger seed agar: brown pigment Cryptococcus neoformans • Cryptococcal antigen test Geotrichum candidum • Geotrichosis – Mimics Candida infections – Infection in debilitated people • Considered a mold • Germ tube: negative • CMT: arthroconidia and true hyphae • Urease: negative Trichosporon beigelii • Causes White Piedra • Germ tube: negative • CMT: arthroconidia, true hyphae, rare blastoconidia • Urease: positive Rhodotorula species • • • • • Saprophytic yeast found in soil Rarely a pathogen Often encapsulated Urease: positive Colony pigmentation: reddish-orange Saccharomyces species • Rarely a pathogen • Ascospores (acid fast positive) Malassezia furfur • Causes Tinea Versicolor • Septicemia in patients receiving intravenous lipid therapy • KOH: clusters of thick-walled, round budding yeast and short, straight or angular mycelial fragments (spaghetti and meatball appearance) • Lipophilic In Summary … • Key characteristics of yeast • Identification methods – Germ tube – CMT – Biochemical tests • Clinically significant yeast – Candida albicans – Cryptococcus neoformans
