Mycology PDF

VMCB 122 MYCOLOGY GIANNE MAY R. GAGAN Department of Veterinary Paraclinical Sciences College of Veterinary Medicine University of the Philippines Los Baños  General Characteristic of Fungi Eukaryotes Non-photosynthetic Strict aerobes Filamentous Optimum...

VMCB 122 MYCOLOGY GIANNE MAY R. GAGAN Department of Veterinary Paraclinical Sciences College of Veterinary Medicine University of the Philippines Los Baños  General Characteristic of Fungi Eukaryotes Non-photosynthetic Strict aerobes Filamentous Optimum growth (20-30°C) Slow-growing on media Mostly saprophytic General Characteristic of Fungi Nucleated Cell wall (Yeast and hyphae) composed of glucans and chitin Plasma membrane Composed of ergosterol as the primary sterol Animals: cholesterol Disease-oriented Classification of Fungi restricted to the progress mostly outermost layers in immunocompromised of the skin, hair, patients and nails affects skin and affects internal DERMATOPHYTOSIS subcutis organs  Morphological Classification of Fungi Molds vs Yeast Multicellular filamentous Oval, spherical, elongated Branching filaments (hyphae) = singular cells 3–5 μm in 2–10 μm, with cross walls or diameter septate (except zygomycetes non- septate) Purely morphological terms, no taxonomic significance Some fungi are dimorphic  Morphological Classification of Fungi Dimorphic fungi Can occur as either as mold (25°C) or yeasts (37°C) https://universe84a.com/wp-content/uploads/2021/01/Blastomycosis-1024x576.jpg  General Structure of Molds (Vegetative) Hyphae- (Aerial) Hyphae – grow penetrate the artificial above the surface to absorb the nutrients. of the artificial medium. Reproductive Hyphae: Mycelium aerial hyphae carrying the reproductive structure (spore). Spores Hyphae Fundamental structural units Long and branched, threadlike filaments Tubular cell wall Grow and secrete enzymes Expand into new food resources Types: 1. Septate: hyphae divided into cells by crosswalls 2. Coenocytic: no crosswalls  Mycelium Tangled (disorganized) mass of hyphae To create a vast feeding network Increase surface area: volume ratio à increase ability to absorb nutrients Spores Individual reproductive bodies of fungi Sporangiospores Ascospores Conidia Basidiospores Arthrospores Zygospores Chlamydospores Oospores Blastospores For most fungi of medical importance, only asexual spores are recognized Asexual Spores Disseminate to other locations and germinate when conditions are favorable The shape color and arrangement of asexual spores aid in the identification of fungi  Sporangiospores Conidiospore/Conidia  Arthrospores Figure: Arthroconidia of Coccidioides. Image Source: CDC.  Chlamydospores Blastospores Candida albicans Candida albicans Nutrition and Growth High humidity, acidic pH (3.8–5.6) High sugar concentration (4–5 %), carbon, phosphorus, sulphur and traces of potassium, magnesium, iron and calcium Peptone in the media and keratin in the skin act as a nitrogen source. Fungi grow slower than bacteria and at a lower temperature and lower pH than most bacteria prefer. Specimen Collection and Transport Fungi are very hardy organisms No special requirement for transport media Sterile container necessary to prevent bacterial contamination Room temperature transport Numerous anatomic sites appropriate for culture Fungal Culture Sabouraud Dextrose Agar (SDA) Selective medium to allow growth of fungi and inhibit growth of bacteria Acidic medium Contains peptone, dextrose, antibiotics (gentamicin, chloramphenicol) and cycloheximide Emmons modification of Sabouraud (neutral pH) Potato Dextrose Agar (PDA) Corn Meal Agar Mycosel agar Mounting Fluids/Stains for Fungi Lactophenol cotton blue (LPCB) used for primary study of fungal morphology isolated from animals and human KOH solution (10 %) Demonstration of dermatophytes from skin scrapings, hair including the hair bulb, nail fragments, sputum and pus India Ink- Cryptococcus Grocott Methenamine Silver-Nitrate (GMS) stain Periodic Acid-Schiff (PAS) stain  Microscopic Appearance: Lactophenol Cotton Blue most widely used mounting medium and staining agent for slide preparation for microscopic examination of fungi Components: Phenol: kills live organisms Glycerol: prevents drying Lactic acid: preserves fungal structures Cotton blue: stains the chitin Microscopic Appearance: KOH wet mount This figure was uploaded by Sangeeta Amladi Figure 1: Skin scraping and KOH mount showing branching fungal hyphae in dermatophyte infection Fig. 1. 10% KOH mount showing broad aseptate hyphae (yellow arrow) along with branched This figure was uploaded by dr. mousumi Paul septate hyphae (blue arrow) (400x magnification).  Microscopic Appearance: India Ink Negative staining of Cryptococcal capsules Image source: Doering lab Fig. 1 India ink stain of Cryptococcus neoformans cells showing extensive capsule development and characteristic budding of daughter cells. Cell diameters excluding capsule are 5-10 um across Available via license: CC BY-NC 4.0 Content may be subject to copyright. Tease Mount Method 1. Prepare an oil-free microscopic slide. 2. Place a drop of Lactophenol Blue on slide. 3. Flame 2 inoculating needles. 4. Pick mycelia then add the specimen over the drop of LPB 5. Gently tease the fungus apart and thinly spread it out. 6. Place a clean coverslip 7. Examine slide under the microscope. Classification of Fungi Relies mostly on morphological criteria Pigmentation ( colorless , pigmented) Surface elevations or depressions Size and appearance after specified incubation time Shape of hyphae ( racquet –shaped, spiral hyphae ) Presence or absence of septa Types of spores  A Gross Examination Growth on SDA Colony characteristics (typography of colony, pigment, surface, texture, size etc.) Methods B Microscopic Examination Staining reaction = lactophenol cotton blue (LPCB) Identification of structures  Yeast Moist, creamy and white in color Unicellular and round to oval Similar in appearance Usually five to ten times larger than to Staphylococcus colonies. bacteria and can be visualized at 400X total magnification.  Yeast reproduce asexually by budding and the newly produced cell, called a bud or blastospore, protrudes from the periphery of the parent cell. Phase-contrast microscopy but also with negative https://www.sciencedirect.com/topics/immunolo staining (the cells are suspended in India ink) gy-and-microbiology/cell-budding (https://archive.bio.ed.ac.uk/jdeacon/microbes/yeast.htm)  Mold Aspergillus Penicillium Rhizopus https://open.maricopa.edu/myfirstbook/chapter/fungi/  Microsporum canis Dermatophytes Microsporum gypseum Trichphyton mentagrophytes Aspergillus niger Aspergillus Aspergillus fumigatus Aspergillus flavuus Candida albicans Pathogenic yeast Malassezia pachydermitis Cryptococcus neoformans Pathogenic Mucor zygomycetes Rhizopus Common Pencillium sp. contaminants  DERMATOPHYTES Molds capable of parasitizing only keratinized epidermal structures: superficial skin, hair, feathers, horn, hooves, claws, and nails Microscopic morphology of the micro and/or macroconidia is the most reliable identification character  Microsporum canis Has septate hyphae, with numerous macroconidia; macroconidia are rather long (10-25 X 35-110 µm), spindle or fusoid in shape Downy to woolly, to fluffy, hairy and silky. Typically it exhibits a light yellowish pigment at the periphery and growth shows closely spaced radial grooves.  Microsporum gypseum Has septate hyphae along which sessile or stalked clavate (club shaped) microconidia; The fusiform (spindle shaped) macroconidia are relatively thin walled, verrucose (with bumpy surface) and contain about 3 – 6 internal cells Colony generally described as yellowish-buff to a dark cream or tan colour in colour; may develop a sterile white ‘feathered’ hyphal border or a cottony white raised center; Colonies are generally flat with a granular or powdery texture  Trichophyton mentagrophytes Produces septate hyphae (show typical spiral hyphae) from which branched conidiophores extend; microconidia: spherical to pyriform; macrocondia: cigar shaped Exhibits moderately rapid growth and matures within 6 – 10 days; downy, powdery or even fluffy texture  ASPERGILLUS Ubiquitous in the environment, often involved with respiratory tract infection Rapidly growing septate fungi Conidiospores are unbranched (helpful in differentiating with Penicillium sp. )  Aspergillus niger Septate, hyphae. Conidiophores (Stipes) are long; Conidia are globose, brown to black in colour starting with a white to yellowish felt-like mat of mycelia, quickly turning black as conida develop the pigment aspergillin during maturation  Aspergillus fumigatus Hyphae are septate with smooth walled conidiophores. Vesicles are subclavate in shape.Conidiogenous cells (phialides) are flask shaped, The surface growth is velvety, downy or powdery, showing various shades of green, most commonly a blue-green to a grey-green with a narrow white border. The colour typically darkens with age.  Aspergillus flavus Septate hyphae with rather long conidiophore Very rapid rate of growth, maturing in about three days. Surface is greenish-yellow to olive and may have a white border. Texture is often floccose, especially near the center and overall can be velvety to woolly. Penicillium vs Aspergillus  PATHOGENIC YEAST Can cause superficial infections, mucosal infections, and invasive diseases in humans and animals. Some common pathogenic yeasts in domestic animals include: Candida, Cryptococcus, Malassezia  Candida albicans Round to oval cells about 4 to 8 µm Grows rapidly in culture, reaching maturity in as little as three days; Colonies are cream coloured, raised, entire, smooth & butyrous Candida albicans can reproduce through budding (asexual reproduction wherein a new organism develops from an outgrowth or bud due to cell division at one particular site) or through spore formation (chlamyspore and blastospore)  Cryptococcus neoformans Colonies producing capsules on culture are often evident by their glistening wet or mucoid appearance whereas colonies which fail to produce capsules or have diminished ability to form capsules typically produce dull, creamy, butyrous colonies.  India ink preparation. Cryptococcus neoformans is spherical, encapsulated, non- myceliated, non-fermenting fungal cell  Malassezia pachydermatis The typical bottle-shaped yeast cells with a wide septum between mother and daughter cells colonies are small, smooth and often have an odor not unlike a “wet-dog”  PATHOGENIC ZYGOMYCETES Opportunistic and often affect immunocompromised people. Saprophytic/Opportunistic fungal pathogens  Rhizopus Fluffy white, grey colored colonies in the beginning which later on becomes yellowish or brownish colonies dotted with black sporangia  Mucor spp. Mucor has broad hyphae which are scarcely or non-septate. Sporangiophores are long, may be branched and terminate in a round spore- filled sporangia (50µm-300µm diameter). rapidly growing fungus which will fill a culture plate in a matter of a few days with a woolly growth resembling cotton candy. New growth is white in colour but turns a greyish-brown with aging. Penicillium sp. Most species exhibit rapid growth and become fully mature in about 5 days. The surface appearance is usually described as velvety to powdery. The colony colour varies with the species but is usually a green, blue-green or grey-green, often with a white edge. Exudates of various colours may also form on the surface. Penicillium sp. Penicillium produces septate, hyaline hyphae, about 1.5 µm to 5 µm in diameter. Conidiophores can be simple or branched depending on the species. Phialides are, to a greater or lesser degree, flask or ampule shaped depending on the species; Conidia can vary in shape from spherical to ovoid to fusiform, again dependent on the species Questions? THANK YOU FOR LISTENING! Email: [email protected]