Mycology Lab2

Microbiology Laboratory: OTOMYCOSES (02/16/2005) Aspergillus spp.

Mucor spp. Flat compact colonies, white at first then becoming black, green, bluish or yellow.

Small, one-celled spores irradiating out from swollen base (see arrows)

Classification: contaminants Characteristic structures: Coenocytic hyphae, sporangium but no rhizoids Mounting fluid used: LPCB Curvularia spp. Classification: contaminants Characteristic structures: dematiaceous hyphae and note the enlargement at the middle of the conidia that makes it curved. Mounting fluid used: LPCB

Classification: contaminants Characteristic structures: hyaline, septate hyphae, presence of foot cell Mounting fluid used: LPCB The opaque white discoloration of the appears similar to white onychomycoses.

Fusarium spp. Classification: contaminants Characteristic structures: banana-shaped septated conidia

nail plate superficial Mounting fluid used: LPCB

Rhizopus spp. Classification: contaminants Characteristic structures: Coenocytic hyphae, sporangium, and rhizophoids Mounting fluid used: Lactophenol cotton blue (LPCB)

The yellow, thickened nail plate with abundant subungual debris has the appearance of a typical examination.

Trichophyton rubrum Cephalosporium Cultured on Sabouraud medium plus 2 antibiotics for 3 weeks at room temperature. Usually fluffy white with red underside. Some strains look granular on the surface.

White to tan to rose-colored colony, eventually developing white aerial hyphae.

Single-celled, clear, elliptical spores held together in a ball unless broken loose.

No large spores. Small spores are spherical to elongate, may be produced directly on hyphae. Similar to Trichophyton metagrophytes but does not penetrate hair in vitro.

Penicillium spp. Classification: contaminants Characteristic structures: Hyaline, septate hyphae Mounting fluid used: LPCB

Actual slide Trichophyton tonsurans

White colony at first but developing blue to green color.

Small, round spores borne in “brushlike” formations.

Cultured on Sabouraud medium plus 2 antibiotics to 3 weeks at room temperature. Powdery red to yellow to brown on surface, wrinkled colony undersurface, may be reddish brown. Yellow variant exists. Many small club-shaped spores, often enlarging to resemble small balloons. Improved growth with thiamine.

Actual slide Phialophora verucosa Fonsecae compacta Surface is dark green to black. The colonies are heaped, brittle with regular indented borders. There are brownish hyphae on the surface. Hyphae are septate, brown, branching and bear Cladosporium type of conidiophores. Outstanding features are cask-like shape of conidia and compact arrangement of conidial chain. Basidiobolus ranarum Colonies are flat, yellowish gray to creamy gray, glabrous becoming radially folded and covered by a fine powder, white surface mycelium.

Disease produced: Chromomycosis (2nd) Characteristic features: dematiaceous hyphae, septate with vase shaped coniodiophores. Mounting fluid use: LPCB Microscopic

Fonsecea pedrosi Disease produced: Chromomycosis Characteristics: Dematiaceous septate hyphae Mounting fluid used: LPCB Hi he

g r magnification Microscopic

There is the presence of large vegetative hyphae forming numerous round, smooth, thickwalled zygospores with 2 closely appressed beaklike appendages. Graphium (asexual state) Pseudoallescherichia boydii (sexual state) The coremia or synnemate (conidial structures) of the Graphium state of P. boydii have terminal hyaline conidia, club-shaped or cylindrical, approx. 6 x 3 um. In the sexual state (P. boydii) is large 50-200 um in diameter, round, brown, cleistothecia are found containing ascopspores.

Sporothrix schenkii Typical appearance in vitro at 25°C on malt extract agar.

The organism is dimorphic fungus. At 26°C, the colony is composed of septate hyphae. Slender conidiophores branch off at right angles and bear clusters.

Microscopic

Scedosporium apiospremum (asexual state) Pseudoallescherichia boydii (sexual state) The Scedosporium type of conidia of P. boydii may rise directly from the septate hyphae of tfrom the tipo of the conidiospores, appear truncated at the base, and sometime resemble the conidia of Blastomyces dermatidis, The hyphae are long and slender, branch at acute angles and thus may resemble aspergilli. Pseudoallescherichia boydii The colonies have a cottony surface that is white to gray-brown in color and gets darker with age of the culture. The reverse is also white turning brown with age. Microscopic (P. boydii)

< White to tan yeast colonies after 1 to 3 weeks in incubation on brain heart infusion agar.

Round, oval, or “cigar-saped” > yeast cells, 1-3 um x 4-10 um Dark, greasy-looking culture after 1-2 weeks incubation on Sabouraud medium

Clear, septate hyphae with spores (3-6um) in “daisy-like” clusters.

Cladosporium carrioni The colony has dark surface, flat with slightly raised center. It is covered with velvety dull gray, gray green or purplish brown, short napped mycelium. Reverse is black. The hyphae are septate, dark with lateral and terminal conidiophores of varying size. The conidiophores produce long, branching chains of brown, smooth walled, oval somewhat pointed conidia which have dark scars of attachment.

Colonies are flat, cream-colored, glabrous becoming radially folded and covered by fine powdery white surface mycelium and conidiophores. The hyphae have few septa. The conidiophores are unbranched forming solitary terminal conidia. The conidia are spherical, single-celled and have a prominent papilla. It may also produce hair-like appendages called villae. Chromomycosis Fission bodies are spherical, dematiaceous structures which neither bud nor produce hyphae; division is by splitting down the middle (i.e fission). Some medical mycologists prefer to call these structure “sclerotic bodies” In this photomicrograph is an example of fission (sclerotic) bodies. These structures are characteristic of the disease known as chromomycosis. There are 2 such structures in this slide (A,B). The most obvious fission body (A) is almost in the middle of the field; it is dematiaceous, does not have any buds and appears to be dividing by fission. This is a photomicrograph of a stained pathology slide. However, the fungal elements in the middle of the slide would be brown even in unstained preparations. Note that this cluster of fungal elements shows neither hyphal formation nor yeast cells. Closer examination of the structures would indicate that they seem to divide by splitting down the middle.

Microscopic

Exophiala (Wangiella) dermatitidis Colonies are slow growing, initially black and yeast-like, becoming suede-like, ovilaceous grey and mould like age.

Exophiala jeanselmei

In new culture, oval and round budding yeast-like cells are formed. Subsequently these cells produce septate hyphae with flask-shaped to cylindrical phialides found at the tip of the phialide and also along the hyphae.

DERMATOPHYTES Microsporum distortum

Microscopic

< Culture on Sabouraud medium plus 2 antibiotcs at room temperature for 3 weeks. Similar to Microsporum canis but with less pigmentation. Large >

SUBCUTANEOUS MYCOSES

but shape.

Conidioblous coronatus

Microsporum audounii

spores similar to Microsporum canis distorted and bent in


5-10 days at room temp. Grows rapidly, producing a cinnamon to brown colored flat colony. Numerous, characteristic, > Large spore; thin-walled, pointed ends with 2-5 septa. Microscopic

No distinguishing spores, > will not grow on rice.

Microsporum vanbreuseghemii walled spores with more than 8 septa.

medium plus 2 at room temp. for 1-3 weeks. White to buff surface: bottom often yellow red brown.

Microsporum nanum Cultured on Sabouraud antibiotics

Egg-shaped, > thin- walled; large spores with 1-3 septa.

Microsporum canis Cultured on Sabouraud medium plus 2 antibiotics at room temp. for 1-2 weeks. White on top with bright yellow underside.

Highly diagnostic large, thickwalled, rough spores containing more than 6 septa.

Microscopic

Malazzezia furfur Spaghetti and meat balls-appearance KOH smear – skin scrapings positive for short hyphal elements oval bodies

with

Microscopic Trichophyton mentagrophytes

Microsporum ferrugineum < Cultured on Sabouraud medium plus 2 antibiotics for 3 weeks at room temp. White to intense orange yellow strains; often sectors. No distinguishing spores. > Prominent septa, giving term “bamboo hyphae”.

Spore formed in grapelike clusters: club-shaped large spores are rare. Often confused with T. rubrum, penetrates hair in vitro. Microscopic

Epidermophyton floccosum Microsporum gypseum
Cultured on Sabouraud medium on 2 antibiotics for 1-3 weeks at room temp. Yellow to green colored surface; green to brown underside.

Large, 2 to 5

club-shaped spores with septa, often form in pairs.

Microscopic

Trichophyton violaceum characteristic features. Rarely see spores. Branched, tangled hyphae. Trichophyton equinum Cultured on Sabouraud medium plus 2 antibiotics at room temp. for 2 weeks. White, fluffy surface with yellow underside: red, orange, and brown colors may develop with age. Small pear-shaped to spherical spores. Large spores rarely seen. Trichophyton concentricum < Cultured on Sabouraud medium plus 2 antibiotics at room temp. for 1-3 weeks. White to orange to brown colony with many wrinkles. Few characteristic > features. Spores among the tangled hyphae: many stimulated by thiamine.

Microscopic _______________________________________________ ~ Leigh ~

Hair penetration Test

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an in vitro test to differentiate T. mentagrophytes and T. rubrum. These 2 isolates are difficult to distinguish between the basis of colony morphology and microscopic appearance.

Hair Baiting Soil Make depressions

Sterile water Half-full Petri dish with sterile hair Subculture on Mycosel Room temp. 2-3 weeks (check water)

Add 5 drops 10% Yeast extract of peptonone Inoculate fungus Trichophyton mentagrophytes or Trichophyton rubrum

Observe for overgrowth (white creamy growth on hair)

Mount on LPCB

Hair Baiting Set-up Incubate at room temperature for 2 weeks

Purpose: to bait the keratophillic fungi (keratin loving fungi) present in soil (geophilic) like Microsporum gypseum Observe: hair strands with whitish material mount using LPCB and observe for M. gypseum

Mount a few pieces of hair in Lactophenol cotton blue (LPCB) Interpretation: (+) hair penetration – T. mentagrophytes (-) hair penetration – T. rubrum Trychophyton rubrum

T. mentagrophytes l e

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filter paper disk (approx. 90 mm in diameter) into the bottom of a standard 100-mm sterile petri dish. Add approx. 15 ml of sterile water. Place into the water a lock of child’s hair that has been sterilized in an autoclave. It is recommended that the hair used not have been recently treated with shampoo or hair sprays. Transfer a portion of the colony to be studied directly into water in the Petri dish. Replace the lid of the Petri dish and incubate at 25°C (room temp.) In approx. 10 to 14 days, place a few hair in a drop of water on a microscope slide, overlay a cover slip, and examine under low and high power magnification of a microscope for the presence of conical shaped penetrations of the hair shaft. Trichophyton mentagrophytes has the ability to invade the hair shaft; Trichophyton rubrum grows on the surface of the hair, but is unable to penetrate.

Slide Culture Method 2 sterile rods are placed at the bottom of a Petri dish on which a sterile glass microscope slide is placed. Blocks or circles of agar are transferred aseptically to the microscope slide. A fragment of the fungal colony to be studied is inoculated onto the sides of the agar, which is coverslipped asepticall and incubated at 25°C. When the colony is mature, the coverslip is removed and both the coverslip and the growth on the slide surface below the agar block are mounted in LPCB and sealed with nail polish or mounting medium.

~ Leigh ~ [email protected]

Photograph of hair shaft infected with Trichophyton mentagrophytes in hair-baiting.