Microbiology Laboratory: OTOMYCOSES (02/16/2005)

Aspergillus 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: contaminantsCharacteristic structures: hyaline, septate hyphae, presence of foot cellMounting fluid used: LPCB

The opaque white discoloration of the nail plate appears similar to superficial white onychomycoses.

Rhizopus spp.

Classification: contaminantsCharacteristic structures: Coenocytic hyphae, sporangium, and rhizophoidsMounting fluid used: Lactophenol cotton blue (LPCB)

Cephalosporium

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.

Penicillium spp.

Classification: contaminantsCharacteristic structures: Hyaline, septate hyphaeMounting fluid used: LPCB

White colony at first but developing blue to green color.

Small, round spores borne in “brush-like” formations.

Mucor spp.

Classification: contaminantsCharacteristic structures: Coenocytic hyphae, sporangium but no rhizoidsMounting fluid used: LPCB

Curvularia spp.Classification: contaminantsCharacteristic structures: dematiaceous hyphae and note the enlargement at the middle of the conidia that makes it curved.

Mounting fluid used: LPCB

Fusarium spp.Classification: contaminantsCharacteristic structures: banana-shaped septated conidia

Mounting fluid used: LPCB

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

Trichophyton rubrum

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.

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.

Actual slide

Trichophyton tonsurans

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

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.

There is the presence of large vegetative hyphae forming numerous round, smooth, thick-walled 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.

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 boydiiThe 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)

Phialophora verucosa

Disease produced: Chromomycosis (2nd)Characteristic features: dematiaceous hyphae, septate with vase shaped coniodiophores.

Mounting fluid use: LPCB

Microscopic

Fonsecea pedrosi

Disease produced: ChromomycosisCharacteristics: Dematiaceous septate hyphaeMounting fluid used: LPCB

Hig her

magnification Microscopic

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

< 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.

Microscopic

Exophiala (Wangiella) dermatitidis

Colonies are slow growing, initially black and yeast-like, becoming suede-like, ovilaceous grey and mould like age.

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.

Microscopic

SUBCUTANEOUS MYCOSES

Conidioblous coronatus

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.

ChromomycosisFission 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.

Exophiala jeanselmei

DERMATOPHYTES

Microsporum distortum

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

Large > spores similar to Microsporum canis

but distorted and bent in shape.

Microsporum audounii

<Cultured on Sabouraud plus 2 antibiotics at room temp. for 2-3 weeks. Fluffy white colony with slight yellow underside.

No distinguishing spores, > will not grow on rice.

Microsporum vanbreuseghemii

<Cultured on Sabouraud medium plus 2 antibiotics for 1-2 weeks at room temp. Cottony white surface, may develop pink to tan coloration; bottom often colorless to yellow.

Huge, long, thick rough- > walled spores with more than 8 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, thick-walled, rough spores containing more than 6 septa.

Microscopic

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”.

Microsporum gypseum

<Cultured on Sabouraud medium plus 2 antibiotics for

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

Microsporum nanumCultured on Sabouraud

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

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

Microscopic

Malazzezia furfur

Spaghetti and meat balls-appearanceKOH smear – skin scrapings positive for short hyphal elements with oval bodies

Trichophyton mentagrophytes

<Cultured on Sabouraud medium plus 2 antibiotics for 2-3 weeks at room temp. Usually white, fluffy on top with yellow on bottom; many cultural variations; some producing brown or red pigmentation on bottom.

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

Microscopic

Epidermophyton floccosum

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

Large, club-shaped spores with 2 to 5 septa, often form in pairs.

Microscopic

Trichophyton violaceum

<Cultured on Sabouraud medium plus 2 antibiotics for 3-5 weeks at room temp. Primarily wrinkle, flat, heaped up colony with intense red-purple pigmentation. Improved growth with thiamine.

Few >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

- 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.

Sterile water Half-fullPetri dish with sterile hair

Add 5 drops 10%

Yeast extract of peptonone

Inoculate fungus Trichophyton mentagrophytes or Trichophyton rubrum

Incubate at room temperature for 2 weeks

Mount a few pieces of hair in Lactophenol cotton blue (LPCB)

Interpretation:(+) hair penetration –

T. mentagrophytes(-) hair penetration – T. rubrum

Trychophyton rubrum T. mentagrophytes

1. Pla ce

a

filter paper disk (approx. 90 mm in diameter) into the bottom of a standard 100-mm sterile petri dish.

2. Add approx. 15 ml of sterile water.3. 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.

4. Transfer a portion of the colony to be studied directly into water in the Petri dish.

5. Replace the lid of the Petri dish and incubate at 25°C (room temp.)

6. 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.

7. Trichophyton mentagrophytes has the ability to invade the hair shaft; Trichophyton rubrum grows on the surface of the hair, but is unable to penetrate.

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

Hair Baiting

SoilMake depressions

Subculture on Mycosel

Room temp. 2-3 weeks (check water)

Observe for overgrowth Mount on LPCB(white creamy growth on hair)

Hair Baiting Set-up

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

Slide Culture Method2 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 ~

ustmedc3@yahoogroups.com