Francis Ian L.

Salaver, RMT, MD
 Definition
• Mykos (mushroom)
• Logus (study)

• Study of fungi which include molds

and yeasts
• Unicellular colorless
oval form
• Form of fungi near
body temperature
Yeast
• Multicellular
• Fuzzy, filamentous,
branching form
• Room temperature
Molds
 Dimorphism

• The ability of some fungi to grow in 2

forms under different environmental
conditions
• Grow as
– Yeast form at 37 C
– Mold form at room temperature
Sporothrix
What is found in the rose = MOLDS
What is seen in tissue?
 General Characteristics
• Eukaryotic organisms
• Exist as parasites or saprophytes
(feed on organic substance or decay)

• Reproduction
• Asexual
• Sexual
Bacterial vs Fungal Cultures

Optimum temperatures
 Answer the following:

• Bacterial cultures are incubated at _________

– A. Room temperature
– B. 37 degree Celsius in the incubator
 Answer the following:

• Bacterial cultures are incubated at _________

– A. Room temperature
– B. 37 degree Celsius in the incubator
 Fungi
• Fungus cultures are incubated either at 30°C.
(86°F.) or at room temperature (22-25°C or 72-
77°F).

• A temperature of 30°C is recommended

because nearly all pathogenic fungi grow
better and more rapidly at this temperature.
Bacterial vs Fungal Cultures

Optimum pH
 Answer the following:

• Bacteria culture medium have pH _____ while

fungal culture medium have acidic pH.
– A. Acidic pH
– B. Neutral pH
– C. Alkaline pH
 Answer the following:

• Bacteria culture medium have pH _____ while

fungal culture medium have acidic pH.
– A. Acidic pH
– B. Neutral pH
– C. Alkaline pH

– Fungi grow best at pH of 4-6; average of 5.0

 Answer the following:

• Bacteria, if placed on a hypotonic solution will

__________ while fungi are resistant to
osmotic pressure.

– A. Shrink
– B. Lyse/Burst
– C. Remain the same
 Answer the following:

• Bacteria, if placed on a hypotonic solution will

__________ while fungi are resistant to
osmotic pressure.

– A. Shrink
– B. Lyse/Burst
– C. Remain the same
 Answer the following:

• Bacteria can be (depending on oxygen

requirement) _________ or ____________
 Answer the following:

• Bacteria can be (depending on oxygen

requirement) aerobic or anaerobic
– Yeast are facultative anaerobes
– Molds are aerobic
 NOTE:
• Bacterial growth greatly depends
on the availability of nitrogen and
moisture

• Fungi need less nitrogen and

moisture content
 Nutritional Requirements

• Fungi are often capable of metabolizing

complex carbohydrates that most bacteria
cannot use for nutrients
– Ex. Lignin (a component of wood)
Harmful effects: Allergy
 Harmful effects: Mycoses
• Infection of human body caused by fungi
Harmful effects: Mycotoxicoses
– Disease cause by the toxin of a fungus
– Aspergillus flavus can produce aflatoxin that
can cause hepatocellular carcinoma
Beneficial effects: Preparation of bread

• Yeasts (Saccharomyces) in preparation of

bread
– Bread = sugar + gluten + yeast cells + water

– Gluten forms the framework of bread

– Yeast converts the sugar into carbon dioxide
35 Pesos = 3 pesos sugar plus CO2
Beneficial effects: Preparation of Vaccine
Beneficial effects: Sources of drugs
• Sources of drugs
– Penicillin
– Cephalosporin
Beneficial effects: Mushrooms can be eaten

• Higher fungi, mostly basidiomycetes may

be eaten directly as mushrooms
Beneficial effects: Mushrooms can be eaten

• Saprophytic fungi in soil produce

degradative enzymes essential for the
biologic recycling of organic matter
 Morphology

• Yeast – unicellular, grows best at 37

degree Celsius

• Molds – multicellular; grows best at

room temperature
 Yeast
• Unicellular growth of
fungi
• Spherical or ellipsoidal
(3 – 15 micra)

• Most reproduce by
budding (forming
blastoconidia); few by
binary fission
Budding
 Yeast
• Most reproduce by
budding (forming
blastoconidia); few by
binary fission

– Buds fail to detach

producing elongated yeast
cells (pseudohyphae)
Pseudohyphae
 Hyphae versus pseudohyphae

• Hyphae have no
constrictions

• Hyphae is seen in molds

• Pseudohhyphae is
demonstrated in yeast
form
Septum not constrictions
 Yeast

• Colonies are pasty, opaque, 0.3 – 5 mm in

diameter
• Most are cream-colored; few species
produce pigments

• May be mistaken as bacterial colonies

 Molds
• Produce multicellular,
filamentous, irregular & dry
colonies

• Consist of branching
cylindrical tubules with
diameter from 2–10 micra,
called hyphae
 Molds
• Hyphae grow to form a filamentous mass
of intertwining strands called a
mycelium
Hyphae: 2 portions

• Reproductive or
aerial portion,
– project above the
surface of the agar
medium and contains
the reproductive
structures such as
spores
Hyphae: 2 portions

• Vegetative portion
or thallus
– grows in or on a
substrate and absorbs
water and nutrients
 Hyphae
• Septate if with cross-
walls which divide the
hyphae into uninucleate
cell-like units
 Hyphae
• Nonseptate or
coenocytic if cross-walls
are absent and appear as
long continuous cells with
many nuclei
– Zygomycetes: Mucor,
Rhizopus, Absidia
Zygomycetes (Coenocytic hyphae)

Sporangium Sporangiospores

Sporangiophore
Sporangium
Sporangiospores

Sporangiophore

Nonseptated hyphae

Rhizoids
 Hyphae

• Hyaline if
fungal structures
are colorless

• Highly refractile
 Hyphae

• Dematiaceous if fungi
produce melanin-like
pigments are dark-colored

• The term dematiaceous applies

to fungi that are dark in color.
Lesion of Dematiaceous should also be
brown colored
 Hyphae
• Dematiaceous
• Agents causing
Chromoblastomycosis
– Phialophora
– Exophiala
– Curvularia
– Alternaria

– Brown CAPE

– EX nya si PHIA, naghanap sya ng

ALTERnative na may CURVes
 Fungal structures: Capsule

• Composed predominantly of polysaccharide

• Capsule of Cryptococcus neoformans has

antiphagocytic properties and is associated with
virulence
– Fungal meningitis among immunocompromised
patients
India ink staining
 India Ink Wet Mount

• Used to identify the capsule of Cryptococcus

neoformans
• CSF is directly examined by adding one drop of India
ink

• Capsule appear as a clear halo against a dark

background (negative staining)
White capsule against a black background
 Cell Wall

• Comprise 15-30 % of the dry weight

of fungus
• Generally thicker in yeasts than in
molds
• Appears highly refractile under light
microscope
 Matching type
• 1. Bacteria • A. Cellulose
• 2. Plants • B. Peptidoglycan
• 3. Fungi • C. No cell wall
• 4. Humans • D. Chitin
 Matching type
• 1. Bacteria • A. Cellulose
• 2. Plants • B. Peptidoglycan
• 3. Fungi • C. No cell wall
• 4. Humans • D. Chitin
Bacterial cell wall
Yeast cell wall
 Cell Wall
• 80% or more is carbohydrate
– Major component:chitin (N-acetylglucosamine)

– Varying amounts of glucan, cellulose and

mannan

• The remaining components are protein and

glycoprotein
 Cell Wall
• Fungal cell wall is poorly stained with routine
Hematoxylin and Eosin
• Fungal cell wall stains
– Periodic acid Schiff
– Methenamine silver stain
– Calcofluor white is a fluorescent stain
– Gram stain is useful for Candida and Cryptococcus
Gram stain is only for bacteria except-
Candida
Must be differentiated from S. aureus
Methenamine silver stain - black
Periodic acid schiff - red
 Fluorescence Test

• Calcofluor white stain

–Calcofluor binds to chitin in cell
wall giving a brilliant
fluorescence
Cell membrane
 Cell Membrane
• Bilayered membrane composed of
several phospholipids

• Contain sterols which are essential for

the viability of fungi
– Principal fungal sterols are ergosterol and
zymosterol
 Cytoplasmic Contents

• Nuclei, mitochondria, ER, storage vacuoles

containing hydrolytic enzymes, ions,
metabolites such as amino acids
Laboratory Identification of
Fungi
 Laboratory Methods
• Microscopic Methods
• Cultivation
• Biochemical tests
• Serologic Test
 Microscopic Methods
• Wet Mounts
– KOH
– Lactophenol cotton blue (LPCB)
– India ink
• Fluorescence Test
• Staining
10% Potassium hydroxide
 KOH (10%) Wet Mount
• Direct preparation from clinical specimen

• OH acts as clearing agent

– Dissolves keratin
– Eliminates debris

• One drop of KOH is added to specimen on a slide

– Allow specimen to clear for 20 minutes or warm slide
gently
• Disadvantage: poor contrast
Lactophenol Cotton Blue
 LPCB Wet Mount

• Lactic acid preserve the fungal structures

• Phenol is a killing agent
• Cotton blue color fungal structure

• Add one drop of LPCB to specimen on a

slide
• Advantage: impart a blue color to cell wall
 Fluorescence Test

• Wood’s lamp (UV light)

– Infected hair and skin will
fluoresce when examined in
the dark
 Cultivation
Requirements
• Culture media must include sources
of
– Nitrogen, Nitrate, Amino acids, Carbon,
Vitamins and minerals
– Antimicrobial supplements
• Cyclohexamide: inhibit contaminating
saprophytic fungi
• Chloramphenicol: inhibit bacterial growth
 Primary Isolation Media
• Sabouraud dextrose agar (SDA)
– for primary isolation of saprophytic and
pathogenic fungi

– SDA-CC for recovery of pathogenic

fungi
• bacteria and saprophytic fungi are
inhibited
But there are fastidious fungi….

Some require specific nutrients

before they will grow on culture
media….
 Primary Isolation Media
• Brain heart infusion media
(BHI)
– Recommended for fastidious fungal
organisms
– Made from pig heart and calf brain
• Blood, Peptone, sulfur, carbon,
nitrogen, vitamins and dextrose are
added to promote growth of fastidious
fungi…
 Primary Isolation Media
• Brain heart infusion media (BHI)
– Can be supplemented with blood, and
antimicrobials

– BHI Agar with Blood, Gentamicin and

Chloramphenicol is used for the selective
isolation of fungi from specimens heavily
contaminated with bacteria
 REVIEW

– Yeast multiply by binary fission, budding,

pseudohyphae
 Reproduction

• Sexual
• Asexual
– Molds reproduce asexually by
• (1)fragmentation of hyphae,
• (2) Producing spores
– Spore - a reproductive particle, usually a single cell,
released by a fungus, alga, or plant that may germinate into
another fungi
 FUNGAL SPORES

10/23/2020 144
 Asexual Reproduction
Types of Spores
• Conidia
– Chlamydospore
– Blastospore/Blastoconidia
• YEAST = small buds
– Arthrospores
– Macro- and microconidia
– Phialides
– Sporangiospore
 Asexual Spores: Conidia
• Chlamydospore

– Thick-walled, resistant,
resting spores

– Produced by rounding up
and enlargement of hyphal
segments
– Candida albicans MOLDS
Candida yeast = blastospores and
pseudohyphae
 Asexual Spores: Conidia

• Blastoconidia
– Develop as daughter cell buds off from
parent cell and is pinched off
– Candida

• Pseudohyphae
– Candida
 Asexual Spores: Conidia
• Arthroconidia
– Formed by
fragmentation of the
septate hyphae into
single rectangular or
barrel-shaped thick-
walled spores
– Coccidioides immitis
 Asexual Spores:
Macroconidia and Microconidia
• Macroconidia
– Large, multiseptate, club,
oval, spindle-shaped
– Cell wall is smooth or
echinulate
• Microcnidia
– Small, unicellular
– Round, elliptical. pyriform,
tear-shaped
 Asexual Spores:
Macroconidia and Microconidia
• Macroconidia
– Large, multiseptate, club,
oval, spindle-shaped
– Cell wall is smooth or
echinulate
• Microconidia
– Small, unicellular
– Round, elliptical. pyriform,
tear-shaped
 Macroconidia and Microconidia
Dermatophytes

• Microsporum
• Trichophyton
• Epidermophyton

– Dermatophytes are fungi that require keratin for

growth. These fungi can cause superficial
infections of the skin, hair, and nails.
 Dermatophytes
• The genus Trichophyton is capable of invading
the hair, skin and nails (All three so tri).

• The genus Epidermophyton involves the skin

and nails only (Not Hair).

• The genus Microsporum involves only the hair

and the skin (Not Nail).
• Microsporum
– Large multicellular
spindle shaped
macroconidia;
microconidia rarely
produced
• Trichophyton
– Predominant forms are
microconidia;
macroconidia
uncommon

– Do not
fluoresce on
Wood’s lamp
• Epidermophyton
– Club-shaped
Macroconidia only;
microconidia not
produced
 Primary Isolation Media
• Dermatophyte test medium
– Recovery of dermatophytes from hair, skin
and nails
– Useful as a screening medium
– Dermatophytes produce alkaline metabolites
which raise the pH and change the color of
medium into RED
 Asexual Reproduction
Types of Spores
• Conidia
– Chlamydospore
– Blastospore
– Arthrospores
– Macro- and microconidia
– Phialides
– Sporangiospore
 Asexual Spores:
Phialides (Secondary Branches)
• Aspergillus
• Penicillium
• Phialophora
• Exophiala (Annelids)

– A phialide/sterigma is an elongated and flask

shaped projection rising from the vesicle in certain
fungal groups
 Aspergillus

• Branching
septate hyphae
which bears
terminal
conidiophore

*
*
 Aspergillus
• Conidiophore
expands to large
inverted flask
shaped vesicle

*
 Aspergillus
• Vesicle is covered
with phialides on
upper half of the
vesicle

*
 Aspergillus
• Phialides contain
rough walled
conidia/
conidiospores

*
*
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 Penicillium
• Septated Hyphae with
conidiophore that exhibit
branching to form metulae

• Brush-like conidiophore
(metulae) give rise to
phialides with conidiospores

*
 Penicillium

• Brush-like
conidiophores
that give rise to
phialides from
which chain of
conidia arise
*
*
*
 Phialophora
• Flask-shaped
phialides with cup-
shaped collarettes
and clusters of
conidia at the end
 Exophiala

• Conidiophores are
long and cylindrical
with tapered tip and
ringed by clusters of
conidia
 Asexual Reproduction
Types of Spores
• Conidia
– Chlamydospore
– Blastospore
– Arthrospores
– Macro- and microconidia
– Phialides
– Sporangiospore
 Asexual Spores:
Sporangiospores
• Asexual spores
contained in a sac-like
or sporangium
• Unique among fungi
with Nonseptated or
coenocytic hyphae
– Zygomycetes
– Rhizopus, Mucor
Absidia
 Mucor
• Nonseptated
hyphae with no
rhizoids
• The
sporangiophores
arise singly with
sac called
sporangium

*
*
 Mucor
• Sporangium
contains
sporangiospores

*
*
 Rhizopus

• Nonseptated hyphae
• With Rhizoids
• Sporangiophores
• Sporangium

*
*
 Asexual Reproduction
Types of Spores
• Conidia
– Chlamydospore
– Blastospore
– Arthrospores
– Macro- and microconidia
– Phialides
– Sporangiospore
Sexual Reproduction
• Teleomorphs
– The sexual stage of a fungus

• Anamorphs
– The asexual stage of a fungus
 Sexual Reproduction
• Requires formation of specialized
structures so that fertilization or nuclear
fusion can occur

• Meiosis occurs with reduction division of 2

fertile cells, followed by merging of the
cells and nuclear fusion
 Sexual Reproduction
• A fungal sexual spore results from sexual
reproduction,
• 3 phases
– Meiosis – diploid nucleus become haploid
– Plasmogamy – fusion of cytoplasm resulting
to one organism with two nuclei
– Karyogamy – fusion of nuclei to form back
diploid state
• Plasmogamy
– A haploid nucleus of a donor cell (+) penetrates
the cytoplasm of a recipient cell (-)

• Karyogamy
– The (+) and (-) nuclei fuse to form a diploid
zygote nucleus

• Meiosis
– The diploid nucleus gives rise to haploid nuclei
(sexual spores)
 Types of Sexual Spores
• Ascospores
– Contained in a sac-like ascus
Note
Analyze this
 Therefore
• Ascomycetes

• Reproduce Sexually by forming

Ascospores
• Reproduce Asexually by forming
Conidiospores/conidia/microconidia/mac
roconidia
 Types of Sexual Spores
• Zygospores
– Fusion of 2 identical cells arising from the
same hypha
 Zygomycota
• Aseptate hyphae
• Asexual spores: sporangiospores
• Sexual spores: zygospores
• Clinically important members:
– Rhizopus
– Mucor
– Absidia
 Types of Sexual Spores

• Basidiospores
– Contained in a club-
shaped basidium

– MUSHROOMS
 Therefore
• Basidiomycetes

• Reproduce Sexually by forming Basidiospores

• Reproduce Asexually by hyphal fragmentation
Imperfect Fungi (Deuteromycetes)
• Those fungi that do not show any sexual stage
• Candida, Cryptococcus, Penicillium