COLLEGE OF PUBLIC HEALTH

UNIVERSITY OF TILE PHILIPPINES MANILA

SEAMED TROPMED Philippines
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This material was prepared by Ms. Mary Ann C..Sison of the Department of Medical
Microbiology, College of Public Health, University of the Philippines Manila solely for the
use of participants enrolled in the 40th Postgraduate Course in Diagnostic Mycology.
Please do not distribute without permission.

Ms. Mary Ann C. Sison, DMM, MPH

University Researcher II
Department of Medical Microbiology, College of Pkiblic Health
University of the Philippines Manila
4 0 ' Postgraduate Course in Diagnostic Mycology. "Superficial and
Cutaneous Fungal Agents Prepared Slides"
March 9, 2021
 STUDY OF SUPERFICIAL FUNGAL AGENTS

Materials:

KOH mounts of Pityriasis versicolor, tinea nigra, black and white piedra lesions

Procedure:

1. Examine these mounts under the microscope.

2. Draw your observations and label them accordingly.

Results: Pityriasis versicolor

Cluster of oval budding cells from

skin scrapings
( “meatballs”)

Short, septated, hyphal elements

(“spaghetti”)

Source:https://mycology.adelaide.edu.au/descriptions/yeasts/malassezia/

Exophiala werneckii
( Hortaea werneckii, Phaeoannellomyces
werneckii)

Oval, two celled yeast like cells from skin

scrapings of the infected lesion of the hand

Photo courtesy of MCSison

 STUDY OF SUPERFICIAL FUNGAL AGENTS

Piedraia hortae 


Chlamydospore

Photo courtesy of MCSison Source: https://www.ijtrichology.com/article.asp?

Hard , black nodules on the hair shaft KOH mount of crushed nodule shows brown
dermatiaceous closely septated hyphae with
few chlamydospores

Trichosporon beigelii

soft greyish-white
nodules along the
hair shaft.
Photo courtesy of MCSison

Source: https://mycology.adelaide.edu.au/mycoses/superficial/
 STUDY OF SUPERFICIAL FUNGAL AGENTS

CULTURAL AND MICROSCOPIC CHARACTERISTICS OF

COMMON SUPERFICIAL FUNGAL AGENTS

Malassezia furfur

DISEASE: Pityriasis versicolor

GROWTH IN-VITRO: Possible only culture medium is overlaid with olive oil or
provided with some other source of fatty acid.

COLONY MORPHOLOGY: Creamy and smooth

Source: http://thunderhouse4-
yuri.blogspot.com/2014/07/malassezia-furfur-
complex.html

MICROSCOPIC MORPHOLOGY:

The colonies contain oval-budding cells with broad-based isthmus. The

yeast cells may be seen as reduced phialides. Elongation to form hyphal
elements is rare.

Source: http://thunderhouse4-
yuri.blogspot.com/2014/07/malassezia-furfur-
complex.html
 STUDY OF SUPERFICIAL FUNGAL AGENTS

Exophiala werneckii (Phaeoannellomyces werneckii)

DISEASE: Tinea nigra

RATE OF GROWTH: slow. Reaches maximum size within 14 days.

COLONY MORPHOLOGY: Young colonies appear moist, shiny-black and

yeastlike. Older colonies have less moisture on the surface and become dark
green with grayish mycelium on the surface.

Source: http://thunderhouse4-yuri.blogspot.com/2014/03/hortaea-werneckii.html

MICROSCOPIC MORPHOLOGY:

Yo u n g c o l o n i e s y i e l d
yeastlike cells,some possessing
cross-walls.Older cultures develop
hyphae that are septate, dark,
crooked, and laterally produce oval
conidia (2-4x4-8 um) that are
colorless or pigmented and one or
two-celled. Conidia may form from
mature yeastlike cells, short
conidiophores, or points
along the hyphae.

Photo courtesy of MCSison

 STUDY OF SUPERFICIAL FUNGAL AGENTS

Piedraia hortae

DISEASE: Black piedra

RATE OF GROWTH: slow, mature in 21 days

COLONY MORPHOLOGY: Appear greenish-brown to black, which is raised or

flat in the center and is glabrous to cerebriform.

Source: https://www.ijtrichology.com/article.asp?

MICROSCOPIC MORPHOLOGY:
Hyphae are dark, thick-walled,
closely septate, with numerous
intercalary chlamydoconidia or
enlarged irregular cells. Asci and
ascospores may be produced in
culture.

Ascus with ascospores

 STUDY OF SUPERFICIAL FUNGAL AGENTS

Trichosporon beigelli

DISEASE: White piedra

RATE OF GROWTH: Rapid. Sensitive to cycloheximide

COLONY MORPHOLOGY: Appears as a cream-colored, slimy, soft colony, later

becoming finely wrinkled, raised in the center, darker, and more firmly attached to
the agar.

Source: https://mycology.adelaide.edu.au/
mycoses/superficial/

MICROSCOPIC MORPHOLOGY:

Hyphae are hyaline and septate

that fragment into oval or rectangular
arthroconidia, 2-4 x 3-9 um. Some
blastoconidia are also seen.
No asci are
produced.
Arthroconidia

Source: http://thunderhouse4-yuri.blogspot.com/2014/08/
trichosporon-species.html
 KOH MOUNT
The most commonly used method for direct examination of clinical
materials for fungal elements involves the use of 10% potassium hydroxide
(KOH) solution. Its advantage is that it leaves the fungal elements intact, while
its alkali nature partially digests the host cells. The procedure is also very simple,
and the test yields rapid results. One can easily understand why this is a
preferred method in the clinical setting.

Materials:

1. 10%KOH
2. Clean glass slide
3. Cover slip
4. Skin scrapings

Procedure:

1. Place a portion of the specimen to be examined on a clean glass slide.

2. Add a drop of 10% KOH

3. Place a cover slip on the preparation and allow to stand at room

temperature for 5 to 10 minutes. You may also pass the slide preparation
over the flame once or twice to hasten the clearing of cellular material.

4. Examine with a microscope under low power and diminished light.

Click the link to view video on KOH mount:

https://youtu.be/REAdCUkmBqM
Interpretation:
Positive : Fungal elements are seen.
Negative: Fungal elements are not seen.

Results: You will be given two (2) clinical specimens. Indicate which is positive
and which is negative. Draw your observations on the space provided.

Specimen no: #1 Interpretation: _________

Specimen no: #2 Interpretation: ________

 CULTURAL AND MICROSCOPIC CHARACTERISTIC
OF DERMATOPHYTES
Genus Microsporum

Attacks the hair and skin only. Microsporum-infected hairs are described
as “small-spored ectothrix” because infected hairs are covered with a sheath of
very small spores. Since these spores are formed by fragmentation of the small
mycelium, their arthropores.

Microsporum gypseum

Colony is moderately fast growing, flat with irregularly fringed border and
coarse, powdery surface, ranging from light ochre to deep cinnamon brown.
Tufts of white, fluffy, sterile growth develop rapidly on surface of colony.

Microscopically, numerous macroconida (fuseaux), ellipsoid and many celled

(2-4) are seen. Macroconicia are rare, clavate, usually sessile on hyphae.
Racquet mycelium, pectinate hyphae and nodule bodies are present.

Photo courtesy of MCSison

 Microsporum canis

Colony is fast growing with abundant cottony mycelium. The surface is at

first white, silky, with bright yellow pigment in peripheral growth. The colony later
becomes buff tan in color and may develop concentric or radial grooves. The
reverse of the colony is bright yellow, becoming dull orange-brown.

http://thunderhouse4-yuri.blogspot.com/2012/02/microsporum-canis.html

Microscopically, abundant macroconidia, 8-15 celled, spindle-shaped, often

terminating in a distict knob and with thick echinulate walls are seen.Microconidia
are few, clavate, usually sessile on the hyphae. Racquet mycelium, pectinate
hyphae and nodular bodies are present. Spiral hyphae are produced on rice
medium.

Photo courtesy of MCSison

 Genus Epidemophyton

Attacks skin and nails. In the tissue, these appear as segmented,

branching mycelial elements identical with Microsporum and Trichophyton.

Epidermophyton flocossum

Colony is slow gowing, at first fluffy white, then becoming velvety or

powdery. Surface is flat or radially folded, tan to olive green; reverse side of
colony yellowish-tan color.

Source: https://microbenotes.com/epidermophyton-floccosum/

Microscopically, characterized by delopment of numerous macroconidia,

2-4 celled, large, broad, blunt to clavate with smooth this walls, which are borne
singly or in groups of 2 to 3. Chlamydospores are numerous.

Photo courtesy of MCSison

Genus Trichophyton

Attacks hair, skin and nails. Infected hairs may show the fungus either
confined to the cortex of the hair (endothrix type) as chairns of spores in parallel
rows, or outside of the hair (ectrothrix type) as chains of small or large spores. In
the skin and nails, these fungi appear as segmented, branching mycelial
elements.

Trichophyton mentagrophytes

Primary cultures vary from granular, light buff to full rose tan in color to
fluffy cotton growth, pure white in color.

http://thunderhouse4-yuri.blogspot.com/2013/04/trichophyton-
mentagrophytes-complex.html

Microscopically, the granular type of colony usually yields numerous

microconidia and spirals and may show macroconidia in small numbers. The
fluffy types show sterile vegetative hyphae with some microconidia. Spirals and
amcroconidia are rare or lacking. In both types the conidia are usually oval or
round and in clusters.

Photo courtesy of MCSison

 Trichopyton rubrum

Primary colonies are fluffy and pure white, later velvety with a deep rose-
purple or reddish-colored reverse. This pigmentation may spread up to the
marginal hyphae of the colony.

http://thunderhouse4-yuri.blogspot.com/2012/02/trichophyton-rubrum.html

Microscopically. Many sterile vegetative hyphae and some microconidia

are present which are arranged “en thyrse” (along side of hyphae). Microconidia
are usually somewhat oblong or rectangular in shape. Macrocondia may be
present.

http://thunderhouse4-yuri.blogspot.com/2012/02/trichophyton-rubrum.html
 Trichophyton tonsurans

Colony is slow growing with compact, creamy-white velvety surface;

becomes folded with deep crater-like depression in the center of the colony.
Variation in color of the colony is common.

http://thunderhouse4-yuri.blogspot.com/2012/09/microsporum-
tonsurans.html

Microscopically, microconidia abundant, many chlamydospores, but

macroconidia are absent or rare. The microconidia are delicate and elongate but
may become large and irregular in size and shape. Macroconidia are clavate or
irregular with thin smooth walls.

Photo courtesy of MCSison

 Exercise 6E
TESTS TO DIFFERENTIATE TRICHOPHYTON
MENTAGROPHYTES
FROM TRICHOPHYTON RUBRUM
A. IN-VITRO HAIR CULTURE

Materials:

1. Cultures (atypical) of T. mentagrophytes and T. rubrum on sabouraud’s

agar

2. Strand of sterilized human hair (1 cm. long)

3. Sterile petri dishes and water blanks

4. 10% sterile yeast extract

Procedure:

1. Place several short strands of sterilized human hair in a sterile petri dish.

2. Add 25 ml of sterile distilled water and 2-3 drops of 10% yeast extract.

3. Inoculate plates with several small fragments of the test organism (1 plate
for T. mentagrophytes and another plate for T. rubrum).

4. Incubate at room temperature and examine the plates at regular intervals

over a period of four (4) weeks.

Results:

1. Make slide mounts in lactophenol cotton blue or KOH of hairs attacked by

the fungus and note the presence of absence of perforations on the hair
shaft. Hair strands exposed to T. mentagrophytes are penetrated radially
by organized groups of hyphae that form wedge-shaped perforations. On
the other hand, T. rubrum does not perforate hair.

https://bit.ly/3efATHq
Questions:

1. From the clinical aspect, why is it very important to determine whether the
infecting fungus is T. mentagrophytes or T. rubrum?

2. What other methods are available in differentiating atypical strains of T.

mentagrophytes and T. rubrum?

B. UREASE TEST

Materials:

1. Urea broth (2)

2. Cultures of T. mentagrophytes and T. rubrum on Sabouraud’s agar

Results:

1. T. mentagrophytes splits urea more rapidly than other dermatophytes. T.

rubrum gives a negtive or a weak positive reaction. What is the color
produced throughout the urea broth for T. mentagrophytes and T. rubrum?

T. rubrum T. mentagrophytes
Urease negative Urease positive

2. After how many days was the color change observed? Why is it important
to note the length of time?