Microbiology Laboratory: OTOMYCOSES (02/16/2005)

Aspergillus spp. Mucor spp.

Flat compact colonies, white at first Classification: contaminants

then becoming black, green, bluish or Characteristic structures:
yellow. Coenocytic hyphae, sporangium
but no rhizoids
Mounting fluid used: LPCB

Small, one-celled spores irradiating out

from swollen base (see arrows) Curvularia spp.
Classification: contaminants
Characteristic structures:
dematiaceous hyphae and
note the enlargement at the
Classification: contaminants middle of the conidia that
Characteristic structures: hyaline, makes it curved.
septate hyphae, presence of foot cell Mounting fluid used: LPCB
Mounting fluid used: LPCB
Fusarium spp.
The opaque white Classification:
discoloration of the nail plate contaminants
appears similar to superficial Characteristic structures:
white banana-shaped septated
onychomycoses. conidia
Mounting fluid used: LPCB

Rhizopus spp.

Classification: contaminants The yellow, thickened nail plate

Characteristic structures: with abundant subungual
Coenocytic hyphae, debris has the appearance of a
sporangium, and rhizophoids typical examination.
Mounting fluid used:
Lactophenol cotton blue (LPCB)

Trichophyton rubrum
Cephalosporium
Cultured on Sabouraud medium
White to tan to rose-colored colony, plus 2 antibiotics for 3 weeks at
eventually developing white aerial room temperature. Usually fluffy
hyphae. white with red underside. Some
strains look granular on the
surface.

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
Penicillium spp. metagrophytes but does not
penetrate hair in vitro.
Classification: contaminants
Characteristic structures:
Hyaline, septate hyphae
Mounting fluid used: LPCB Actual slide

Trichophyton tonsurans

White colony at first but developing Cultured on Sabouraud medium

blue to green color. plus 2 antibiotics to 3 weeks at room
temperature. Powdery red to yellow
to brown on surface, wrinkled colony
undersurface, may be reddish
Small, round spores borne in “brush- brown. Yellow variant exists.
like” formations.

Many small club-shaped spores,

often enlarging to resemble small
balloons. Improved growth with
thiamine.
 Actual slide

Phialophora verucosa

Disease produced: Chromomycosis (2nd)

Fonsecae compacta
Surface is dark green to black. The
colonies are heaped, brittle with
regular indented borders. There are
brownish hyphae on the surface.
Characteristic features: dematiaceous hyphae, septate with
vase shaped coniodiophores.
Mounting fluid use: LPCB
Microscopic

Hyphae are septate, brown,

branching and bear Cladosporium
type of conidiophores. Outstanding Fonsecea pedrosi
features are cask-like shape of
conidia and compact arrangement of Disease produced:
conidial chain. Chromomycosis
Characteristics: Dematiaceous
septate hyphae
Basidiobolus ranarum Mounting fluid used: LPCB

Colonies are flat, yellowish gray to

creamy gray, glabrous becoming Hi
radially folded and covered by a fine g he
powder, white surface mycelium. r

magnification
Microscopic

There is the presence of large Sporothrix schenkii

vegetative hyphae forming
numerous round, smooth, thick-
walled zygospores with 2 closely Typical appearance in vitro at
appressed beaklike appendages. 25°C on malt extract agar.

Graphium (asexual state)

Pseudoallescherichia boydii (sexual state)
The coremia or synnemate
(conidial structures) of the
Graphium state of P. boydii The organism is dimorphic fungus. At
have terminal hyaline 26°C, the colony is composed of septate
conidia, club-shaped or hyphae. Slender conidiophores branch
cylindrical, approx. 6 x 3 off at right angles and bear clusters.
um.

In the sexual state (P. boydii) is large 50-200 um in

diameter, round, brown, cleistothecia are found containing
ascopspores. 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. < White to tan yeast colonies after 1 to 3 weeks in
incubation on brain heart infusion agar.
Microscopic (P. boydii)
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.
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.
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 nanum
Cultured 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-appearance
KOH 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. Hair Baiting
- These 2 isolates are difficult to distinguish
between the basis of colony morphology and Soil
microscopic appearance. Make depressions

Sterile water Half-full

Petri dish with sterile hair

Subculture on
Mycosel
Add 5 drops 10% Room temp. 2-3 weeks
Yeast extract of (check water)
peptonone

Inoculate fungus
Trichophyton Observe for overgrowth Mount on LPCB
mentagrophytes (white creamy growth on hair)
or Trichophyton
rubrum
Hair Baiting Set-up
Incubate at room temperature for 2 Purpose: to bait the
weeks keratophillic fungi
(keratin loving fungi)
Mount a few pieces of present in soil
hair in Lactophenol (geophilic) like
cotton blue (LPCB) Microsporum gypseum
Observe: hair strands
Interpretation: with whitish material
(+) hair penetration – T. mount using LPCB and
mentagrophytes observe for M. gypseum
(-) hair penetration – T. rubrum

Trychophyton rubrum T. mentagrophytes

1.P
l ac
e a

Slide Culture Method

filter paper disk 2 sterile rods are placed
(approx. 90 mm in diameter) into the bottom of a at the bottom of a Petri
standard 100-mm sterile petri dish. dish on which a sterile
2. Add approx. 15 ml of sterile water. glass microscope slide
3. Place into the water a lock of child’s hair that has is placed. Blocks or
been sterilized in an autoclave. It is recommended circles of agar are
that the hair used not have been recently treated transferred aseptically
with shampoo or hair sprays. to the microscope slide.
4. Transfer a portion of the colony to be studied A fragment of the fungal
directly into water in the Petri dish. colony to be studied is
5. Replace the lid of the Petri dish and incubate at inoculated onto the sides of the agar, which is
25°C (room temp.) coverslipped asepticall and incubated at 25°C. When
6. In approx. 10 to 14 days, place a few hair in a the colony is mature, the coverslip is removed and
drop of water on a microscope slide, overlay a both the coverslip and the growth on the slide surface
cover slip, and examine under low and high power below the agar block are mounted in LPCB and sealed
magnification of a microscope for the presence of with nail polish or mounting medium.
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 ~ Leigh ~
penetrate.
ustmedc3@yahoogroups.com

Photograph of hair shaft

infected with Trichophyton
mentagrophytes in hair-baiting.