Lorma Colleges

College of Medical Laboratory Science

MYCOVIRO
❖ Survive at optimal temp. of
35-37oC (body temp.).
INTRODUCTION TO ❖ Thrive mostly in vivo or
inside the body.
MYCOLOGY ❖ E.g.: Candida albicans
(candidiasis or moniliasis)
Mr. Charlie Cruz ➢ Filamentous molds
❖ Agents of diseases:
o Superficial skin
TOPIC OUTLINE infection (cutaneous
mycosis)
I. Introduction o Disseminated deep-
i. Common Terms in Medical seated visceral
Mycology disease (systemic
ii. Overview of Fungi mycosis)
II. General Characteristics Histoplasmosis
i. Shared Characteristics Chromoblastom
III. Epidemiology ycosis
IV. Yeasts Cryptococcosis
❖ Survive at room temp.
V. Molds
❖ Filamentous in appearance
i. Morphology
due to hyphae.
VI. Hyaline versus Phaeoid o Group of hyphae:
VII. Dimorphism and Polymorphism mycelium
VIII. Reproduction • True pathogens: historically pathogenic fungi
IX. Taxonomy • Opportunistic pathogens: saprophytic fungi
i. Mucorales (thrive on dead bodies, scavengers)
ii. Ascomycota ➢ Causes invasive infections such as in:
iii. Basidiomycota ❖ Immunocompromised host
X. Fungi Imperfecta ❖ Immunosuppressive
XI. Fungal Organisms infection: HIV/ADIS
❖ High-dose cancer
chemotherapy
INTRODUCTION ❖ Solid organ transplantation
➢ E.g.: Pneumocystis jirovecii
Mycology
• Study of fungi Common Terms in Medical Mycology
• Alongside taxonomy, environmental impact,
and genetic and biochemical properties (used Term Definition
traditionally to identify fungal species).
Aerial mycelium Hyphae produced above
the surface of the agar
Medical mycology
media
• Study of medically-significant fungi
Anamorph Asexual form of fungal
• Study of fungi and their relationship to
sporulation (imperfect
human disease.
state)
• Species of fungi that can cause infection or
Arthroconidium Conidium derived from
diseases (mycosis or mycoses) to humans as
the fragmentation of
hosts.
specialized hyphae
➢ Superficial skin infections (cutaneous
Ascocarp (ascoma) Fruiting structure that
mycoses)
contains asci (several
➢ Subcutaneous mycoses
types exist)
➢ Systemic mycoses
• Encompasses:
➢ Single-celled yeasts

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MYCOVIRO
Ascospore Sexual spore formed Holomorph Whole fungus; the
within an ascus anamorphic plus the
following meiosis teleomorphic state of
Ascus (pl., asci) Saclike structure that the fungus
contains ascospores, Homothallic Sexual reproduction can
characteristic of take place within one
Ascomycetes thallus
Basidiospore Sexual spore formed as Hyaline Clear, colorless, or
on an outgrowth of a transparent
basidium Hypha (pl., hyphae) Septate or aseptate
Basidium Structure that contains vegetative unit of a
basidiospores (e.g., mold
mushroom) Intercalary Borne within a hypha
Blastoconidium (pl., Asexual spore formed Macroconidium Larger of two types of
blastoconidia) by budding of the conidia produced by a
hyphal, pseudohyphal, mold
or yeast cell Microconidium Smaller of two types of
Chlamydospore Thick-walled resting or conidia produced by a
survival structure (also mold
called chlamydoconidia) Mold Filamentous fungus that
Cleistothecium (pl., Enclosed ascocarp, reproduces by sexual or
cleistothecia) composed of layers of asexual means
hyphae that contain Mycelium Mass of hyphae that
randomly dispersed asci make up the thallus of a
Collarette Funnel-shaped mold
structure at the apex of Perithecium Enclosed ascocarp with
a phialide a pore at the top
Columella An extension of the through which the
sporangiophore into the ascospores are
base of the sporangium discharged
Conidiogenous cell Cell that produces Phialide Cell with opening
conidia through which conidia
Conidiophore Specialized hyphal are produced
structure that carries Pseudohyphae Connected yeast cells
the conidia (blastoconidia) that
Conidium (pl., conidia) Asexual reproductive resemble a hypha but
structure that forms on contain areas of
the side or the end of a constriction between
hypha or conidiophore adjacent cells
Dematiaceous Pigmented conidia, Sclerotic body (pl., Thick-walled,
(phaeoid) spores, or hyphae due sclerotia) dematiaceous,
to the presence of muriform, rounded cells
melanin found in tissue
Dimorphic Displaying two diagnostic for
morphologic types, one chromoblastomycosis
environmental (mold) Septum (pl., septa) Cross-wall in a hypha
and one in vivo (yeast) Spherule A round structure
Glabrous Smooth, referring to containing endospores
colonial morphology characteristic of
Heterothallic Sexual reproduction Coccidioides species in
requiring the interaction tissue
of two different thalli Sporangiophore Stalk bearing the
(mating strains) sporangium

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MYCOVIRO
Sporangiospore Asexual spore produced GENERAL CHARACTERISTICS
within a sporangium
Sporangium Saclike structure in • Eukaryotic (having true nuclei)
which the asexual • Most fungi are saprophytic (live on dead
sporangiospores organic matter).
develop • Yeasts (moist) and/or molds (filamentous)
Sterigmata Slender outgrowth of a • Dimorphic
cell bearing conidia ➢ Organisms that display both yeast
Teleomorph Sexual form of a fungal and mold forms.
sporulation (perfect • Thermally dimorphic when dependent on
state) temperature.
Terminal Borne at the end of a • Polymorphic
hypha ➢ Fungi having more than one form or
Thallus (pl., thalli) Vegetative growth of a stage.
fungus; includes an • Thick cell walls
interwoven mass of ➢ Made of chitin (protein molecule).
hyphae ➢ Allow the absorbing of specialized
Thermophillic Fungi that grow at a dyes used in identification (ID).
high temperature ❖ Staining properties
Vegetative mycelium Hyphae produced on ❖ Determine what dyes are
the surface or extending being absorb by the fungal
into the agar media cell or organism.
Vesicle Enlarged or swollen cell, • Mostly obligate aerobes (die in the absence
often at the end of a of oxygen)
conidiophore or • Prefer neutral pH
sporangiophore, which
may also be within Shared Characteristics
hyphae
Yeast Single-cell fungus that • Chitin in the cell wall.
reproduces by budding • Ergosterol (lipid) in the cell membrane.
or by fission • Reproduction by means of spores sexually or
Zygospore A mucoraceous spore asexually
formed sexually by the • Lack of chlorophyll (green pigment in plants)
union of two similar • Lack of susceptibility to antibacterial agents
cells
EPIDEMIOLOGY
Overview of Fungi
• Increased fungal infections worldwide
• Diverse group of organisms.
➢ Immunocompromised patients
• Classified as molds (filamentous) and yeasts
➢ Use of antifungal medications
(moist).
❖ Improper use can lead to
• Categories:
drug resistance.
➢ Classic pathogens
• >135,00 valid species
➢ Environmental saprobes (living on
➢ 1000-1500 newly identified annually
nonliving material)
• Implementation of molecular technology will
• May cause a range of signs and symptoms.
continue to impact the evolution of the field
➢ Mild to life-threatening.
of mycology.
➢ Significant in immunocompromised
patients.
YEASTS

• Single, vegetative cells

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MYCOVIRO
➢ Form smooth, creamy, bacterial-like Pseudohyphae are distinguished from true hyphae
colonies without aerial hyphae by the presence of a constriction at the junction of
(branching; septate or aseptate). adjacent cells and by the restriction in size of the
➢ Obtain nutrition from sources daughter cell to that of the parent.
• ID is primarily based on biochemical testing
and molecular diagnostic methods.
• Reproduction via budding or fission

Formation of blastoconidia in yeasts.

Chains of elongated budding yeasts of Candida albicans

producing pseudohyphae observed in sputum. (Gram stain,
1000×)

Budding and nonbudding yeast cells detected in a blood culture

bottle sample. (Gram stain, 100x)
MOLDS
Septum
• Seals the boundary between the daughter • Filamentous mycelium
and parent cells. ➢ Long strands of tube-like structures
called hyphae.
Pseudohypha • Reproduction by spores: asexually or sexually
• Forms if separation does not occur. ➢ Vegetative mycelium
❖ Arthrospores,
chlamydospores,
blastospores
❖ Rhizoids
➢ Aerial mycelium
❖ Can see visibly or
macroscopically
❖ Conidia, sporangia

Aerial mycelia give mold the “woolly” appearance. Vegetative

mycelia are responsible for absorbing nutrients from the
Yeast pseudohyphae of yeast. Filamentous extensions from the
medium.
periphery of colonies of Candida albicans are known colloquially
as “feet.”

Extensions: pseudohyphae

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MYCOVIRO
Morphology Aspergillus spp.
• Hyaline molds
Hyphae • Narrow hyphae that branch at acute angles
• Shape (dichotomous)
• Septate

A, Specialized structures formed in vegetative mycelia by certain

fungal species. B, Rhizopus spp., showing rhizoids (unstained, Characteristic wide, nonseptate hypha of a mucormycete in lung
×200). tissue. (Gomori's methenamine silver stain, 400x)

• Septate The width of the hyphae and angle of branching are

➢ Frequent perpendicular cross-walls important clues to fungal identity.
(septa)
• Sparsely septate Mucormycetes
➢ Frequent perpendicular cross-walls • Also called as Zygomycetes
(septa) • Members of the order Mucorales such as
➢ Limited compared to septate hyphae Rhizopus species
• Broad, ribbon-like hyphae that have
haphazard branching.
• No septum

HYALINE VERSUS PHAEOID

A, Phaeoacremonium sp. displaying septate hyphae (unstained,
×200). B, Mucorales hyphae in tissue appears sparsely septate
Hyaline
(Gomori methenamine silver stain, ×400). • Nonpigmented
• Lightly pigmented

Phaeoid (dematiaceous)
• Darkly pigmented
➢ Melanin production in cell wall

Characteristic narrow branching, septate hyphae of a hyaline

mold in tissue. (Gomori's methenamine silver stain, 400x)

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MYCOVIRO
➢ Generally yeast first moving to mold
as culture ages
❖ E.g.: Exophiala spp.

REPRODUCTION

Asexual reproduction
• Conidia (conidium) following mitosis
➢ Fruiting structures
❖ Phialoconidia
❖ Annellide ring structures
• Phialides
Bipolaris sp. is an example of a phaeoid fungus. Note the dark ➢ Vase-like structures that produce
pigmentation, which is caused by the presence of melanin in phialoconidia
the cell wall (unstained, ×200). • Annellides
➢ Ringed structured that produce
annelloconidia

Powdery surface of the colonies of Aspergillus fumigatus

overlies white masses of mycelium. Adjacent bacterial colonies
contrast with the filamentous fungus. (Sheep blood agar) An example of asexual reproduction is the production of
phialoconidia. Conidia are formed from conidiogenous cells
such as phialide (a vaselike structure). Phialoconidia are
Filamentous nature of mold
“blown out” of the phialide.
• Gives colonies woolly, fluffy, or velvety
appearance
• Sometimes punctuated with a granular or
powdery aspect that is produced by the
formation of a sexual reproductive structure

DIMORPHISM AND POLYMORPHISM

Dimorphism
• Ability to exist in two forms based on growth Arthroconidia, another form of asexual reproduction, are
conditions formed by fragmentation of fertile hyphae.
➢ Mold phase: 22-25oC (room temp.)
➢ Yeast phase: 37oC with increased
carbon dioxide

Polymorphism
• The ability to form yeast and mold in same
culture.

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MYCOVIRO

Fruiting head of Aspergillus fumigatus. The conidiophore is

swollen at the tip to form a vesicle, and phialides arise from
the upper half of the vesicle with chains of conidia present that
align parallel to the long axis of conidiophore. (Lactophenol
cotton blue stain, 400x)
Sexual reproduction occurs by the fusion of compatible nuclei
and subsequent production of a zygospore.

TAXONOMY

Nomenclature
• Historically, fungi have carried multiple
names (genera and species) associated with
reproductive forms.
➢ Obsolete: The International
Botanical Congress (July 2011)
adopted a one fungus, one name
Arthroconidia of Coccidioides spp. Alternating barrel-shaped policy published in the International
arthroconidia are separated by thin-walled, empty disjunctor Code of Nomenclature Article 59.
cells within portions of the hyphae. (Lactophenol cotton blue ➢ International Code of Nomenclature
stain, 400x) for Algae, Fungi, and Plants (January
1, 2013)
Sexual reproduction
❖ Previously called
• Teleomorph of the thallus or holomorph International Code of
Botanical Nomenclature
Anamorph
• Occasionally reproduce asexually Taxonomy of Clinically Significant Fungi
• The causative agents of clinical infections are
Synanamorphs
found in four groups of fungi.
• Name for amorphic strains when more than ➢ Phylum Ascomycota
one anamorph is present for the same ❖ Asexual reproduction by
teleomorph. conidia
• Different asexual forms in the same fungus. ❖ Sexual reproduction by
ascospores
➢ Phylum Basidiomycota

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MYCOVIRO
❖ Sexual reproduction via
basidiospores (on basidia)
➢ Subphylum Mucoromycotina
➢ Subphylum Mucoromycotina and
Entomophythoromycotina
❖ Replaced phyla Zygomycota
❖ Asexual reproduction by
sporangiospores
❖ Sexual reproduction by
zygospores
➢ Form division fungi Imperfecti
(Deuteromycota)
Sporangiophores or Rhizopus spp. Supporting sporangia that
Mucorales contain sporangiospores. Rhizoids arise from the hyphae near
the origin of the sporangiospores. (Lactophenol cotton blue
• Most significant order stain, 100x)
• Rapid growers
• Produce gray to white aerial mycelium with Asexual spores called sporangiospores are produced
hyaline, sparsely septate hyphae by cleavage within an encompassing structure called
• Opportunistic pathogen in the a sporangium.
immunocompromised
• Asexual reproduction Ascomycota
➢ Sporangiophores and
sporangiospores Sexual spores
❖ Results in zygospores (not • Ascospores
seen in clinical laboratories) ➢ Contained in a saclike structure
• E.g. (members): Mucor, Rhizopus, Absidia (ascus)

Species
• Microsporum spp.
• Trichohyton spp.
• Scedosporium boydii

Asexual reproduction by Mucorales is characterized by the

production of spores (sporangiospores) from within a
sporangium.

Ascospores with asci (pale staining) along with budding yeast

cells of Saccharomyces cerevisiae. (Gram stain, 1000x)

The naked asci of the S. cerevisiae resemble oval

yeast cells in which 1-4 individual haploid ascospores
are enclosed.

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MYCOVIRO

Cleistothecium of Scedosporium boydii. This sexual

Elongated macroconidia of Trichophyton mentagrophytes
reproductive structure is specifically referred to as an ascus
along with microconidia are lined up along the surface of
containing ascospores. (Lactophenol cotton blue stain, 400x)
the hypha. (Lactophenol cotton blue stain, 400x)
Cleistothecium
The thallic conidia of dermatophytes (affect the skin)
• Sexual, fruiting body (or an ascocarp) in
are separated by size into two types:
which the ascospores are entirely enclosed by
• Large, separate macroconidia
the cleitothecial wall.
• Small, one-celled microconidia
• Once erupted, it could release ascospores.
➢ Simpler structures

Basidiomycota

• Only pathogen
➢ Filobasidiella neoformans
❖ Sexual form of Cryptococcus
neoformans var. neoformans
• Other genera associated with human
infections
➢ Malassezia (e.g.: M. furfur),
Trichosporon
• ID
➢ Clamp connections at septations

Macroconidia of Epidermophyton floccosum. Typical blunt-

ended macroconidia occur in aggregates along the hyphae
with the absence of microconidia. (Lactophenol cotton blue
stain, 400x)

Cryptococcus neoformans. (India ink stain)

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MYCOVIRO

Malassezia skin disease (pityriasis versicolor)

Image
• From a 42-year-old female patient
• Patient had relapsing disease for the past 6
years.

One characteristic of fungal infections

• Relapsing or they keep on coming back if left
untreated.

FUNGI IMPERFECTA

• Contains the largest number of causative

agents of mycoses.
• Organisms are placed in this group when no
mode of sexual reproduction has been
identified.
• These organisms are identified on the basis
of characteristic asexual reproductive
structures.
• E.g.: Ulocladium, Alternaria tenuis

PARAFUNGAL ORGANISMS

• Organisms that resemble yeast and

protozoans
• Remain unculturable
• Atypical agents of human infections

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MYCOVIRO
Enclosed electric incinerator
• Eliminates the hazards of open gas flames
SPECIMEN COLLECTION,
PROCESSING, AND Use of Petri dishes is hazardous; use screw-top tubes
to prevent aerosols.
LABORATORY DIAGNOSIS
OF FUNGI SPECIMEN COLLECTION, HANDLING, AND
TRANSPORT
Mr. Charlie Cruz, RMT, ASCP
Common specimen types
• Respiratory secretions, hair, skin, nails,
TOPIC OUTLINE tissue, blood, bone marrow, CSF

I. Laboratory Safety Issues Use antimicrobial-containing media to inhibit bacterial

II. Specimen Collection, Handling, and growth.
Transport
1. Hair
III. Predominant Culture Sites for Recovery
➢ A Wood lamp emits UV light >365 nm
of Etiologic Agents
and can be useful in identifying
IV. Major Clinical Syndromes and
infected hairs.
Commonly Associated Fungal Pathogens ➢ The Wood light can be shown on the
V. Guideline for Identification of Fungal scalp.
Isolates ➢ Sterile forceps should be used to pull
VI. Direct Examinaiton of Specimens affected hair.
VII. Staining Characteristics of Fungi ➢ Hairs are placed directly into a sterile
VIII. Isolation Methods Petri Dish then inoculated onto
i. Summary of Primary Fungal fungal medium and incubated at 22
Culture Media to 30° C.
IX. Fungal Identification
i. Immunodiagnosis of Fungal 2. Skins and nails
➢ Area is cleaned with 70% alcohol
Disease
before sampling.
ii. Molecular Identification
➢ Outer edge of a surface legion is
X. Identification of Yeast scraped (skin); nails are cut or
i. Miscellaneous Tests for Yeasts removed.
XI. Identification of Molds ➢ A KOH wet mount is prepared with
i. Miscellaneous Tests for Molds some of the sample collected.
XII. Antifungal Susceptibility ❖ KOH breaks down tissue
i. Antifungal Agents making it easier to view
ii. Antifungal Susceptibility Testing fungal hyphae.
➢ Sample material is inoculated directly
onto the agar.
LABORATORY SAFETY ISSUES
3. Blood and bone marrow
Class II biosafety cabinet ➢ Lysis centrifugation system
• Reduces personal exposure against fungal ❖ Release organisms from cells
conidia ➢ Other systems

Class III biosafety cabinet (risk group 3) 4. CSF (and other sterile fluids)
• Coccidioides immitis/posadasii ➢ Concentrate via centrifugation before
• H. capsulatum inoculation.

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MYCOVIRO
➢ One drop for India ink or MAJOR CLINICAL SYNDROMES AND
cryptococcal antigen test for C. COMMONLY ASSOCIATED FUNGAL
neoformans PATHOGENS
➢ Plated directly (sterile)

5. Abscess, wound, or tissue

➢ Mince and grind tissue
➢ Plate directly or perform other tests

6. Respiratory specimens
➢ Plated directly or digested with
mucolytic prior to plating

7. Urogenital or fecal specimens

➢ Culture (yeast will grow on routine
culture media)
➢ If urine, first voided morning urine
❖ Centrifuge to sediment

PREDOMINANT CULTURE SITES FOR

RECOVERY OF ETIOLOGIC AGENTS

Bon
Respir Blo e Tis Sk Mu Bo
Infection atory od Mar sue in cus ne
row
Blastomycosi
+ + +
s GUIDELINE FOR IDENTIFICATION OF FUNGAL
Histoplasmos ISOLATES
+ + + +
is
Coccidioidom
+ + +
ycosis
Paracoccidioi
+ + +
domycosis
Sporotrichosi
+ + + +
s
Chromoblast
+ +
omycosis
Eumycotic
+ +
mycetoma
Phaeohypho
+ +
mycosis
Organisms may be recovered from multiple sites in
disseminated infections.

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MYCOVIRO
DIRECT EXAMINATIONS OF SPECIMENS

KOH preparation
• 10% to 20% solution of KOH
➢ Some may contain dimethyl sulfoxide
(DMSO) and a stain
• Equal parts sample to KOH
• Cover-slipped and heated gently then cooled C. neoformans: India ink preparation at (a) low & (b) high
for 15 minutes magnification show spherical, narrow-budding yeasts with thick
capsule.
• Purpose of KOH
➢ Dissolve the keratin and skin layers
to visualize dermatophytes (hair,
skin, nails, tissues)

KOH with calcofluor white

• Dye binds to cellulose or polysaccharides
present in chitin.
➢ Apple-green or blue-white in UV light

India ink
• Black background stain
➢ Visualize capsules (looks like halos)
of yeast or bacteria
• Presumptive ID of C. neoformans in CSF but Bone marrow stained with Giemsa stain.
low sensitivity

Tissue
stains STAINING CHARACTERISTICS OF FUNGI
• Periodic acid–Schiff (PAS)
• Gomori methenamine-silver (GMS) Color of
• Hematoxylin and eosin (H&E) Background
Stain Fungal
• Giemsa Color
Element
➢ Detection of H. capsulatum in blood Periodic acid–
or bone marrow Magenta Pink or green
Schiff
• Fontana-Masson Gomori
methenamine Black Green
silver
Purple-to-blue
yeast with
Giemsa Pink to purple
clear halo
(capsule)
Yeast with
India ink clear halo Black
(calsule)
KOH Refractile Clear
KOH–
calcofluor Fluorescent Dark
Fungal hyphae stained with calcofluor white, viewed under UV white
light. Masson-
Brown Pink to purple
Fontana

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MYCOVIRO
ISOLATION METHODS The yeast form of
dimorphic fungi and
Culture media other organisms grow.
SDA
• Sabouraud dextrose agar (SDA) (with or
without antimicrobials) Dermatophytes grow
• Potato dextrose agar (PDA) poorly.
• Brain–heart infusion (BHI) agar enriched Yeasts, such as
with blood and antimicrobials Cryptococcus, grow
• Plates or tubed media well.
➢ Tubes safer to handle and less
susceptible to drying The yeast form of
BHI agar with blood Histoplasma capsulatum
Incubation takes up some of the
• Room temperature or 30° C heme pigment in the
➢ Dimorphic at 37° C medium and becomes
• Maintain 4 to 6 weeks. light tan, with a grainy,
• Examine twice a week for growth. wrinkled texture.
• Incubation characteristics should be *The antibiotics generally used are cycloheximide and
recorded. chloramphenicol.

Summary of Primary Fungal Culture Media Fungal

Principle Purpose
medium
Expected Growth Acid pH & high
Medium
Results dextrose
At 22oC concentration
General-
Initial isolation of inhibits
purpose
pathogens and Sabouraud bacterial
medium for
saprobes. dextrose agar growth but
cultivation or
SDA (SDA) permits growth
isolation of
Dimorphic fungi may of fungi; some
many fungi.
exhibit their mycelial formulations
phase. also contain
Saprobes generally antibiotics.
inhibited on this Nutrient-rich
medium. medium
containing Selective
SDA with antibiotics*
Dermatophytes and chloramphenico isolation of
most of the fungi Inhibitory l & sometimes fungi from
considered primary mold agar gentamicin or specimens that
pathogens grow. (IMA) ciprofloxacin; may contain
Initial isolation of chloramphenico commensal
BHI agar pathogens and l suppresses bacterial flora.
saprobes. the growth of
BHI agar with Recovery of pathogenic many bacteria.
antibiotics fungi.
Initial isolation of
Inhibitory mold agar pathogens except
dermatophytes.
Primary recovery of
Cycloheximide
dermatophytes.
At 37oC

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MYCOVIRO
It is frequently
The
used to target
nonselective
dermatophytes
Nutrient-rich formulation is a
or thermally
medium general-
dimorphic
containing purpose
fungi.
brain/meat medium used
infusion, in the Presumptive ID
peptome & cultivation or for C. albicans
Chloramphenol
dextrose. isolation of
inhibits
bacteria, yeasts C. albicans:
bacteria.
& molds. light to medium
Brain heart green colonies
Chromogenic
infusion agar When CHROMagar
mix selects for
(BHI) supplemeted C. tropicalis:
yeasts &
with dark blue
differentiated
chloramphenic colonies
by color
ol &
formation.
Chloramphenic gentamicin, it is C. kusei: flat,
ol & gentamicin used for pink colonies
can be added selective
for selectivity. isolation of Specialized fungal culture media
fungi from Medium Purpose
specimens that Promotion of characteristic
may contain reproductive structures &
commensal pigmentation useful for
bacterial flora. Cornmeal or potato morphologic identification of
Cycloheximide Selective dextrose agar mold isolates, when general
inhibits the isolation of purpose media prove
growth of many slow-growing inadequate for a particular
saphrophytic pathogenic isolate.
fungi, while fungi that may Promotion of characteristic
permitting be overgown structures (e.g.:
growth of most by rapidly chlamydospores,
(but not all) growing Cornmeal or rice arthroconidia) useful for
pathogenic saprophytic agar with Tween 80 morphologic identification of
fungi. fungi. yeast isolates, when more
Notably, routine identification
Cycloheximide
cycloheximide methods prove inadequate.
-containing
also inhibits the Sabouraud
media Isolation/cultivation of
growth of dextrose, agar,
certain Malssezia spp., all of which
Dixon medium, or
Chloramphenic pathogenic (except M. pachydermatis)
Leeming-Notman
ol & gentamicin fungi including require lipid
medium overlaid
can be added to C. neofromans, supplementation for growth.
with sterile olive oil
inhibit bacteria. many Candida Differentiation between
spp., species of Trichophyton,
Aspergillus spp. Trichophyton agars which can be difficult to
& the speciate based on
zygomycetes, morphology alone.
among others. Bird seed (Niger Demonstration of phenol
seed) agar oxidase activity in

Mycology and Virology 5 [Buen, M. G. V.]

 Lorma Colleges

College of Medical Laboratory Science

MYCOVIRO
Cryptococcus neoformans Topography
isolates. • Verrucose
➢ Furrowed or convoluted
• Umbonate
FUNGAL IDENTIFICATION ➢ Slightly raised in the center
• Rugose
• It typically takes a group of tests to ➢ Furrows radiate out from the center
sufficiently identify fungi.
• Typically start by determining if the isolate is
a yeast or a mold.
➢ Molds do not always produce
structures to facilitate ID.
• Molecular and proteomic tests can be used
with traditional procedures.
➢ DNA hybridization, MALDI-TOF, PCR

Macroscopic
• Colony growth time
• Color
• Texture
• Pigment on reverse of colony

Texture
• Cottony
➢ Loose, high aerial mycelium
• Velvety
Microscopic
➢ Low aerial mycelium
• Septate versus sparsely septate hyphae
• Glabrous
• Hyaline or dematiaceous hyphae
➢ Smooth with no aerial mycelium
• Fruiting structures
• Granular
• Types, size, shape, and arrangement of
➢ Dense and powdery
conidia
• Wooly
➢ High aerial mycelium that is slightly
Tease mount
matted down
• Remove part of colony
• Mount in drop of lactophenol aniline (cotton)
blue
• Tease apart the colony (teasing needle) and
overlay with cover slip to disperse
• Examine at ×100 or ×400

Cellophane tape preparation

• Press cellophane tape gently but firmly to
surface of the colony.
• Press one side of tape to slide with drop of
LPCB.
• Stretch tape across slide, lowering it into the
stain.
• Pull taught and affix other side of tape,
avoiding air bubbles.

Mycology and Virology 6 [Buen, M. G. V.]

 Lorma Colleges

College of Medical Laboratory Science

MYCOVIRO
Scedosporium species
Aspergillus species
Calmodulin Fusarium species
Scedosporium species
Chitin synthase Dermatophyte species
Cytochrome b Candida albicans
Pneumocystis jirovecii
Dihydrofolate reductase
(carinii)
Elongation factor (EF-
Fusarium species
1α gene)
Mitochondrial rRNA Pneumocystis jirovecii
rDNA complex (5S Candida albicans
gene) Pneumocystis jirovecii
rDNA complex (18S
Multiple
Slide culture gene)
• Mostly used to create permanent slides rDNA complex (28S Aspergillus species
• Grow fungus in plug of cornmeal or potato gene) Candida species
dextrose agar with cover slip on top. rDNA complex (IGS
Aspergillus species
• LPCB stain the cover slip after growth gene)
➢ Preserve slide with clear nail polish rDNA complex (ITS
Multiple
around cover slip if desired gene)
RNA polymerase (RPB1
Fusarium species
and RPB2)
IGS: intergenic spacer region
ITS: internal transcribed spacer region
rDNA: ribosomal DNA

IDENTIFICATION OF YEAST

• Colony morphology
• Microscopic morphology
• Physiologic studies
Immunodiagnosis of Fungal Disease • Chromogenic agars
• Rapid commercial identification tests or
Skin test reactivity to fungal antigens panels
• Uses antigen extract • Nucleic acid based methods
• Only valuable if patient has a history of a • MALDI-TOF MS
nonreactive skin test

Antibody detection assays

• Most common is probably double-
immunodiffusion
• Individuals can have detectable antibodies
but not active infection.

Cross-reactivity and interlaboratory discrepancies

have been reported.

Molecular Identification

Gene Target Fungal Species

Actin Candida albicans
β-Tubulin Aspergillus species

Mycology and Virology 7 [Buen, M. G. V.]

 Lorma Colleges

College of Medical Laboratory Science

MYCOVIRO
Miscellaneous Tests for Yeasts

Germ tube production

• Most important and easiest to perform
• Steps:
1. Aseptically transfer several colonies
of yeast to a 12-mm x 75-mm test
tube containing approximately o.5
mL of serum (human, fetal calf,
bovine, or rabbit).
2. Incubate the tube at 35oC for up to 3
hours.
3. Place 1 drop of the mixture on a clean
glass slide and coverslip. Germ tubes have extended from the yeast cells of Candida
4. Examine under high dry (400x) albicans. No constriction is seen at the junction of yeast cell
magnification and reduced light for and germ tube. The walls of the germ tube are parallel. The
yeast was incubated for 2 hours at 37oC in serum. (Gram
the presence of germ tubes.
stain, 400x)

Schematic Diagram Showing Germ Tube Test to Presumptively

Identify Yeasts
C. albicans of cornmeal agar.

(left) Germ tube production, (right) pseudogerm tube

production

Pseudohyphae

Carbohydrate assimilation
• Relies on the aerobic utilization of carbon or
nitrogen as the sole source for the metabolic
process.

Mycology and Virology 8 [Buen, M. G. V.]

 Lorma Colleges

College of Medical Laboratory Science

MYCOVIRO
Chromogenic substrates ➢ Combines nuclear magnetic
• Based on different colors resonance spectroscopy with PCR
➢ Qualitative molecular diagnostic
Cornmeal agar method that can detect and speciate
• For recognition of blastoconidia, the 5 most common Candida spp.;
chlamydoconidia, pseudohyphae, namely, Candida albicans, C.
arthroconidia glabrata, C. parapsilosis, C.
tropicalis, and C. krusei, in
Potassium nitrate assimilation approximately 5 hours
• Positive turns the medium blue.
• Negative result turns the medium yellow. Cryptococcal antigen (CrAg)
• Potassium Nitrate Assimilation Agar contains • Indicator of infection, in serum (a component
yeast carbon base, which provides amino of blood) and in cerebrospinal fluid (CSF).
acids, vitamins, trace elements, and salts, • Detects CrAg in serum a median of 22 days
necessary to support the growth of yeast. before symptoms of meningitis develop

Temperature studies IDENTIFICATION OF MOLDS

Urease • Growth rate

• Christensen urea agar • Colony morphology
• Cryptococcus neoformans, Coccidioides • Microscopic morphology
immitis, Histoplasma capsulatum, Sporothrix • MALDI-TOF MS
schenckii, and species of Trichosporon and
Aspergillus

(1-3)-β-D-glucan detection (cell wall

component)
• Chromogenic test
• Detected in serum during invasive fungal
infections (IFI): invasive aspergillosis,
invasive candidiasis, & P. jirovecii

Galactomannan
• Component of the Aspergillus cell wall
• Diagnosis of invasive pulmonary aspergillosis
• ELISA assay has been developed.

T2 Magnetic Resonance (T2MR) assay

• For diagnosis of invasive candidiasis

Mycology and Virology 9 [Buen, M. G. V.]

 Lorma Colleges

College of Medical Laboratory Science

MYCOVIRO
Miscellanous Tests for Molds Antifungal Susceptibility Testing

• Hair perforation test Clinical and Laboratory Standards Institute (CLSI)

standards
• M27-A3 for yeast testing, M38-A2 for mold
testing, M44-A for disk diffusion testing for
yeasts, and M51-A
➢ Remains limited

• Urease test
• Thiamine requirement
• Trichophyton agars
• Growth on rice grains
• Germ tube production
➢ Most important and easiest test to
perform
• Carbohydrate assimilation
➢ Relies on the aerobic utilization of
carbon or nitrogen as the sole source
for the metabolic process.
• Chromogenic substrates
➢ Based on different colors
• Cornmeal agar
➢ For recognition of blastoconidia,
chlamydoconidia, pseudohyphae,
arthroconidia

ANTIFUNGAL SUSCEPTIBILITY

Antifungal agents

• Main agent is amphotericin B.

➢ Many side effects
❖ Fever, rigors, renal
impairment, and possibly
shock
• Examples of other agents
➢ Fluconazole (FLU), itraconazole
(ITRA), posaconazole (POSA), and
voriconazole (VORI)

Misuse has resulted in the development of

resistance, most notably with C. glabrata.

Mycology and Virology 10 [Buen, M. G. V.]

Mycoses

Charlie Cruz
Course Instructor

1
 Mycoses
 Diseases caused by fungi (singular mycosis)
 Disease categories
 Superficial, cutaneous, subcutaneous, systemic
 Systemic
• Infections not involving the skin or deeper tissues just under
the skin
 Superficial and cutaneous mycoses
• Fungi degrade keratin (dermatophytes)
• Fungi that cannot degrade keratin (nondermatophytes)

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Diagram of Skin and Tissue Layers in
Which Fungal Infections Can Occur

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 Superficial Mycoses
 Superficial mycoses
 Confined to outermost layer of skin or hair
 Tinea or dermatophytoses
• Ringworm
 Tinea versicolor (pityriasis versicolor)
• Discoloration or depigmentation and scaling of the
skin
 Malassezia furfur complex
 Tinea nigra - nondermatophytic
 Piedra - superficial fungal infection of hair shafts

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 Cutaneous Mycoses
 Affect skin, hair, and nails
 Itching, scaling, ringlike patches of skin
 Thickened, discolored nails
 Brittle broken hairs
 Example members
 Trichophyton, Epidermophyton, Microsporum

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Various Forms of Dermatophytoses and
the Respective Affected Sites

6
 Subcutaneous Mycoses
 Involve deeper skin  Clinical features
layers  Chromoblastomycosis
 Muscle, connective • Nonhealing ulcers
tissue, bone  Eumycotic mycetoma
 Generally • Draining sinus tracts
dissemination through  Lymphocutaneous
the blood does not infection that may
occur. become systemic
• Sporothrix schenckii

7
 Systemic Mycoses
 Infections that affect internal organs or deep tissues
of the body
 Frequently enter via the lungs, then spread
 Symptoms
 Fever, fatigue, sometimes chronic cough and chest pain
 Examples
 Histoplasma, Coccidioides, Blastomyces, Aspergillus,
Fusarium, Bipolaris, Candida, Cryptococcus

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 Agents of Superficial Mycoses

Clinically Significant Species

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 General Characteristics
 Affect only the cornified layers (stratum
corneum) of the epidermis
 Affected patients do not show any overt
symptoms.
 Fungal agents do not activate any tissue response of
inflammatory reaction.
 Medical attention usually sought to address
cosmetic concerns

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 Malassezia spp.

 Basidiomycota
 More than 15 species
 Found on the skin of humans and animals
 Many species are lipophilic; the commonly
isolated include:
 M. furfur
 M. globose
 M. obtuse
 M. pachydermatis
 M. sloofiae
 M. sympodialis Copyright © 2015Elsevier
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 Disease: Malassezia
Malassezia furfur
 Patchy lesions or scaling of varying pigmentation
(tinea versicolor)
 Usually on chest, trunk, abdomen
 Brownish scaly lesions on light-skinned persons
 Light lesions on dark-skinned persons
 Causes a disseminated infection in infants and
young children
 Fungemia in immunocompromised patients
 May be cause of dandruff

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Disease: Malassezia
Malassezia furfur

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Disease: Malassezia
Malassezia furfur

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 Diagnosis of Malassezia furfur

 Examine using Skin scrapings in potassium

hydroxide (KOH) preparation
 Yellow fluorescence with Wood lamp
 Spaghetti and meatballs microscopic morphology

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Photograph of “Spaghetti and Meatballs”

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Diagnosis of Malassezia furfur

17
 Disease: Malassezia
Malassezia pachydermatis
 Recovered from skin lesions
 Fungemia in immunocompromised patients

18
 Diagnosis of Malassezia spp.

 Stains
 Examination of skin scrapings
 Oval or bottle-shaped cells with monopolar budding
 “Spaghetti and meatballs” hyphal arrangement
 Cultivation
 Requires agar medium enriched with long chain fatty acids
 Small colonies; creamy white to off-white
 Identification
 Growth on overlaid agar
 “Bowling pin” or “pop bottle” morphologic feature
 Can be identified using substrate utilization profiles

19
Diagnosis of Malassezia spp.

20
 Piedraia hortae
 Causative agent of black piedra
 Infection that occurs in the hairs of the scalp
• Black gritty nodules
 Endemic in tropical Africa, Asia, and Latin
America
 Examine infected hairs are removed and placed
in 10% to 20% KOH.
 Nodules may be crushed to reveal asci.

21
 Disease: Black Piedra
 clinical presentation: multiple 1–2 mm hard,
darkly pigmented oval nodules adherent to
hair shafts
 nodules are composed of aligned,
dichotomously branching hyphae surrounding a
cement-filled stroma with areas containing asci,
each of which holds eight fusiform, curved
ascoconidia.

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Disease: Black Piedra

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 Trichosporon spp.

 The genus Trichosporon, has been

reorganized into three genera.
 Trichosporon spp.
 Apiotrichum spp.
 Cutaneotrichosporon spp.

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 Trichosporon spp.

 Extremely variable
 White piedra on hair shaft
 Surrounding hair of scalp, face, and pubic region
 Opportunistic pathogen
 White piedra is endemic in tropical areas of South
America, Africa, and parts of Asia

25
 Disease: Trichosporon Group

 Causes trichosporonosis
 Disseminated trichosporonosis (most common)
 Skin lesions
 Endocarditis
 Endophthalmitis
 Brain abscess
 Respiratory infections
 May cause white piedra in patients who are
immunocompetent

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Disease: Trichosporon Group

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Diagnosis of Trichosporon Group

 Stains
 Hyaline hyphae
 Numerous round to rectangular arthroconidia
 Few blastoconidia
 Grow rapidly on primary media
 Smooth colonies, cream-colored, heaped, dry-to-
moist wrinkled and yeastlike
• Some wrinkling upon maturation
 Some may appear white, dry, and powdery
 Absence of carbohydrate fermentation
 Utilization of potassium nitrate, urease positive

28
Diagnosis of Trichosporon Group
(continued)
 Identification
 Presence of rectangular arthroconidia with round
ends
 Presence of septate hyaline hyphae
 Urease positive
 Substrate utilization profile

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Diagnosis of Trichosporon Group
(continued)

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Lactophenol Cotton Blue (LPCB) Preparation
Showing Blastoconidia and Arthroconidia

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 Hortaea werneckii
 Formerly Phaeoannellomyces/Exophiala werneckii
 Tinea nigra
 Brown to black nonscaly macules
• Palms of hands and soles of feet
 No inflammation or reaction to fungus
 Skin scraping in 10% to 20% KOH
 Septate hyphae and budding cells
 Culture
 Shiny, moist, yeastlike colonies, brown to olive or greenish-
black
 Endemic in tropical areas
 Central and South America, Africa, and Asia

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Tinea nigra

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Tinea nigra

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 Hortaea werneckii
 Colonies are slow growing, initially mucoid,
yeast-like and shiny-black
 Microscopically, colonies consist of brown to
dark olivaceous, septate hyphal elements and
numerous two-celled, pale brown, cylindrical
to spindle-shaped yeast-like cells that taper
towards the ends to form an annellide.
 Most yeast-like cells also have prominent
darkly-pigmented septa.

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 Hortaea werneckii
 Annellides may also arise from the hyphae.
 Conidia are one to two-celled, cylindrical to
spindle-shaped, hyaline to pale brown and
usually occur in aggregated masses.
 Chlamydospores also present.

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 Hortaea werneckii
 Key features:
Hyphomycete, two-celled yeast-like cells
producing annelloconidia.
 Molecular identification:
An ITS-primer specific for H. werneckii was
developed by Abliz et al. (2003). ITS
sequencing can also assist identification.

37
Hortaea werneckii

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Microscopic Structures of H. werneckii

39
Slide 1

Agents of Cutaneous Mycoses

Clinically Significant
Species

Charlie Cruz
Course Instructor

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Elsevier Inc. 1
Slide 2

General Characteristics
 Grow on hair, nails (onychomycosis), and
cutaneous skin layers with keratin
➢ Commonly referred to as dermatophytes
• Example: athlete’s foot (Tinea pedis)
 Etiologic agents
➢ Trichophyton (hair, skin, and nails)
➢ Microsporum (hair and skin)
➢ Epidermophyton (skin and nails)

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Slide 3

General Characteristics
 Two sizes of reproductive cells—important in
species ID
➢ Macroconidium and microconidium
 Some members also have teleomorphic
stages.

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Slide 4

Types of Infections
 Infections involving hair
➢ Affects different body sites with different symptoms
➢ Scalp infections most severe
 Infections involving the nails
➢ Onychomycosis
 Tinea pedis (athlete’s foot)
 When disease progresses around the side of
the foot from the sole
➢ Moccasin foot

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Slide 5

Ringworm
 Disease—Causes cutaneous mycoses
➢ Tinea corporis—Ringworm of the body
➢ Tinea cruris—Ringworm of the groin
➢ Tinea capitis—Ringworm of the scalp
➢ Tinea barbae—Ringworm of the beard
➢ Tinea unguium—Ringworm of the nail
➢ Tinea pedis—Ringworm of the feet

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Slide 6

Tinea corporis

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Tinea corporis (Waist and inframammary area). Tinea corporis is a superficial fungal
skin infection of the body caused by dermatophytes. Tinea corporis can be found
worldwide. It is specifically defined by the location of the lesions that may involve the
trunk, neck, arms, and legs.
Slide 7

Tinea cruris

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Tinea cruris also commonly known as jock itch, is a surface (superficial) fungal infection
that affects the skin of your genitals, inner thighs and buttocks. Tinea cruris is most
often seen in adult men. Tinea cruris causes an itchy, red, often ring-shaped rash in
these warm, moist areas of your body.
Slide 8

Tinea capitis

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Tinea capitis, also known as ringworm or herpes tonsurans infection, is a fungal

infection of the scalp hair. It is caused primarily by the dermatophyte species
Microsporum and Trichophyton. The fungi can penetrate the hair follicle's outer root
sheath and ultimately may invade the hair shaft. Clinically, tinea capitis can be divided
into inflammatory and non-inflammatory types. The non-inflammatory type usually will
not be complicated by scarring alopecia. The inflammatory type may result in a kerion, a
painful nodule with pus, and scarring alopecia.
Slide 9

Tinea barbae

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Tinea barbae is a superficial fungal infection of the skin, hair, and hair follicles caused
by dermatophytes. Though dermatophyte infections are one of the most common skin
infections in humans, tinea barbae is relatively rare. Lack of awareness about the
disease in the medical community often leads to missed diagnosis and
mismanagement.
Slide 10

Tinea unguium

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Onychomycosis is a fungal infection of the nail unit. When onychomycosis is caused by

dermatophytes, it is called tinea unguium. The term onychomycosis encompasses not
only the dermatophytes but the yeasts and saprophytic molds infections as well. An
abnormal nail that is not caused by a fungal infection is a type of dystrophic nail. The
most frequent cause of onychomycosis is Trichophyton rubrum, but other
dermatophytes, including Trichophyton
mentagrophytes and Epidermophyton floccosum, can be caused as well. The
dermatophytes are identified in 90% of the toenail and 50% of fingernail
onychomycosis. Candida albicans accounts for 2% of onychomycosis, occurring
especially in fingernails.
Slide 11

Tinea pedis

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Athlete’s foot, or tinea pedis, is an infection of the skin and feet that can be caused by a
variety of different fungi. Although tinea pedis can affect any portion of the foot, the
infection most often affects the space between the toes. Athlete’s foot is typically
characterized by skin fissures or scales that can be red and itchy.
Slide 12

Systemic Dermatophyte Infections

 Immunocompromised
 In disseminated disease
➢ Nodules with varying size to granulomas possible
 Primarily in kidney transplant patients and is
thought to have spread from athlete’s foot or
onychomycosis

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Slide 13

Epidermophyton floccosum
 Colony morphology
➢ Yellow to yellow tan with feathered edges; small in
diameter
 Microscopically
➢ Only one size of conidia—known as macroconidia
• Club-shaped macroconidia often clustered in groups
• Three to five cells with thin smooth walls
• Look like beaver tails
 Distributed worldwide

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Slide 14

Culture of Epidermophyton
floccosum

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Colonies are usually slow growing, greenish-brown or khaki-coloured with a suede-like

surface, raised and folded in the centre, with a flat periphery and submerged fringe of
growth. Older cultures may develop white pleomorphic tufts of mycelium. A deep
yellowish-brown reverse pigment is usually present.
Slide 15

Macroconidia and chlamydoconidia

of E. floccosum

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Microscopic morphology shows characteristic smooth, thin-walled macroconidia which

are often produced in clusters growing directly from the hyphae. Numerous
chlamydospores are formed in older cultures. Microconidia are not formed.
Slide 16

Microsporum
 Large, spindle-shaped, rough-walled
macroconidia
 Thick walls with four or more septa
 Small and club-shaped microconidia
 Cultures that produce aerial hyphae
➢ Texture—Velvety, powdery, glabrous, or cottony
➢ Color—Whitish, buff to cinnamon brown

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Slide 17

Microsporum canis
 Causes ringworm in children and adults
 Is spread by infected animals
 Infected hair shafts fluoresce yellow-green
under a Wood’s lamp

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Slide 18

Microsporum canis (continued)

 Colony
➢ Initially white, fluffy, silky to granular with a
characteristic lemon-yellow fringe at periphery
➢ Reverse side is bright yellow
 Microscopically
➢ Spindle-shaped, multicelled macroconidia
➢ Pointed and slightly turned at one tip
➢ Thick spiny (echinulate) wall with cross-septations
➢ Scattered microconidia laterally from hyphae

 Worldwide distribution

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Slide 19

Macroconidia of M. canis

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Microsporum canis showing spindle-shaped, echinulate macroconidia with thick walls

and tapered ends, which are key features in identification of this species (unstained,
×450).
Slide 20

Microsporum gypseum
 Colony morphology
➢ Flat, initially white, turning light brown to reddish-
brown on maturity
➢ Sugary or granular appearance
 Microscopically
➢ Fusiform, moderately thick-walled conidia
➢ Up to six cells
➢ Some isolates have thin, filamentous distal end tail
 Found in soils worldwide

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Slide 21

Macroconidia of M. gypseum

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Microsporum gypseum showing fusiform, moderately thick-walled macroconidia

containing several cells (lactophenol cotton blue preparation, ×450).
Slide 22

Microsporum audouinii
 Slow-growing anthropomorphic dermatophyte
➢ Used to be leading cause of tinea capitis among
school children
 Spread by infected hair
 Infected hair shafts fluoresce yellow-green under
a Wood’s lamp

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Slide 23

Microsporum audouinii (continued)

 Rare conidia
➢ Some isolates produce terminal chlamydoconidium-
like swellings.
 Colonies
➢ Cottony and white with little to no pigment on reverse
➢ Produce velvety aerial mycelium that is colorless to
light gray
➢ Reverse side: appears salmon-pink to reddish brown

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Slide 24

Culture of Microsporum audouinii

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Colonies are flat, spreading, greyish-white to light tan-white in colour, and have a dense
suede-like to downy surface, suggestive of mouse fur in texture. Reverse can be yellow-
brown to reddish-brown in colour. Some strains may show no reverse pigment.
Slide 25

Chlamydoconidium of Microsporum
audouinii

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Macroconidia and microconidia are rarely produced, most cultures are sterile or produce
only occasional thick-walled terminal or intercalary chlamydospores. When present,
macroconidia may resemble those of M. canis but are usually longer, smoother and
more irregularly fusiform in shape; microconidia, when present, are pyriform to clavate
in shape.
Slide 26

Growth on rice grains:

Microsporum audouinii

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(left) Microsporum audouinii showing poor growth on rice grains, usually being visible
only as a brown discolouration and (right) Microsporum canis on rice grains showing
good growth, yellow pigmentation and sporulation.
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Trichophyton
 General characteristics
➢ Is widely distributed
➢ Responsible for tinea corporis, tinea capitis, tinea
unguium, and tinea barbae
➢ Infected hairs do not fluoresce under Wood’s lamp
(ultraviolet [UV])
 Invasion
➢ Endothrix—Hair shaft is filled with arthroconidia
➢ Ectothrix—Spores are found around the hair shaft

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Slide 28

Trichophyton (continued)
 Identification
➢ Macroconidia—Smooth, club-shaped, and thin-
walled
➢ Microconidia—Spherical, pyriform (teardrop), or
clavate (club-like)

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Slide 29

Trichophyton mentagrophytes
 Perforates hair
 Rapid growing
 Has a rose-brown reverse surface
 Macroscopically
➢ Colonies: downy to granular based on number of
microconidia present

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Slide 30

Trichophyton mentagrophytes
(continued)
 Microscopically
➢ Microconidia form loose grapelike clusters (en
grappe).
• Globose to tear shaped
➢ Macroconidia present in very low numbers or
absent.
• Thin-walled, smooth-walled, cigar-shaped four to five
cells separated by parallel cross-walls (2-5 septa),
narrow attachment to base
 Worldwide distribution

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Slide 31

Example of Globose, Tear-Drop

Microconidia

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Trichophyton mentagrophytes showing globose, teardrop-shaped microconidia

(Nomarski optics, ×450).
Slide 32

T. mentagrophytes

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A, Trichophyton mentagrophytes showing numerous microconidia in grapelike clusters. B,

Several thin-walled macroconidia also are present (500).
Slide 33

Trichophyton rubrum
 Does not perforate hair
 Slow growing
 Has a cherry-red reverse surface
 Colony morphology
➢ Initially white with cottony, fluffy, velvety, or
granular colonies
➢ Key observation
• Water-soluble wine-red pigment on the reverse of the
colony

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Slide 34

Trichophyton rubrum (continued)

 Microscopically
➢ Microconidia are tiny, clavate or peg shaped, and
distributed on either side of the hyphal strands.
• “Birds on a fence”
➢ Macroconidia similar to T. mentagrophytes
• Thin-walled
• Smooth-walled
• Multicelled
• Elongated and pencil shaped or cigar-shaped (3-8 septa)
• Rare

 Worldwide distribution

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Slide 35

Microconidia of T. rubrum

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Trichophyton rubrum showing clavate- or peg-shaped microconidia (lactophenol cotton

blue preparation, ×450).
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Microconidia of T. rubrum

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Trichophyton rubrum showing numerous pyriform microconidia borne singly on hyphae (750).
Slide 37

Trichophyton tonsurans
 Colony morphology
➢ Rust-colored pigment on colony’s reverse when
grown on Sabrouad dextrose agar
 Microscopic
➢ Microconidia extremely variable in shape
• Ranges from round to peg shapes
 Leading cause of tinea capitis in children in
many parts of the world (including the United
States)

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Slide 38

Trichophyton tonsurans cultures

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T. tonsurans cultures show considerable variation in texture and colour. The colour may
vary from pale-buff to yellow to dark-brown. The reverse colour varies from yellow-
brown to reddish-brown to deep mahogany.

Colonies show considerable variation in texture and colour. They may be suede-like to
powdery, flat with a raised centre or folded, often with radial grooves. The colour may
vary from pale-buff to yellow, (the sulfureum form which resembles Epidermophyton
floccosum), to dark-brown. The reverse colour varies from yellow-brown to reddish-
brown to deep mahogany. Hyphae are relatively broad, irregular, much branched with
numerous septa.
Slide 39

Trichophyton tonsurans
microconidia and macroconidia

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Numerous characteristic microconidia varying in size and shape from long clavate to
broad pyriform, are borne at right angles to the hyphae, which often remain unstained
by lactophenol cotton blue. Very occasional smooth, thin-walled, irregular, clavate
macroconidia may be present on some cultures. Numerous swollen giant forms of
microconidia and chlamydospores are produced in older cultures.