MYCOLOGY

Medical Microbiology (Laboratory)

BS Biology 4-5 | Dr. Delos Reyes | 1st Sem 2023-2024

I. MYCOLOGY II. FUNGAL STRUCTURES

A. TERMINOLOGY A. HYPHAE

● Long, branching filaments that form the mycelium.

Molds Multicellular fungi
● Septate hyphae
Yeasts Single-cell fungi ○ Have cellular separation or cross-walls.
○ Range diameter from 3 to 6 um.
Mycosis Fungal infection ● Sparsely septate (formerly aseptate) hyphae
○ Contain few if any cellular separations.
Systemic mycosis Multiorgan infection caused by fungi ○ Range in diameter from 5 to 15 um.
○ Coenocytic
Opportunistic Fungal disease that occurs primarily ■ Refers to hyphae lacking cross-walls.
mycosis in immunocompromised ● Pseudohyphae
patients ○ Chain of cells formed by budding that resemble
true hyphae
Dimorphic fungi Fungi that show both a non-mold ○ Differ from true hyphae because they are
(e.g., yeast) and mold phase constricted at the septa
■ Form branches that begin with septation
Saprobe Organism capable of living on ■ Have terminal cells smaller than other cells
decaying organic material

B. MOLD VS YEAST

Molds Yeast

Growth Multicellular Unicellular

pattern filamentous colonies opaque colonies ● Vegetative hyphae
○ Function in food absorption
Microscopic Branching cylindric Spherical to ○ The portion that extends below the agar surface or
appearance tubules (hyphae) ellipsoid nutrient substrate.
● Aerial hyphae
Size 2-10 um 3-15 um ○ Extend above the agar or nutrient substrate
(diameter) ○ Their function is to support reproductive structures
called conidia.
Features Septa (cross walls)

Positive: septated
Negative: coenocytic

Identification Ontogeny and conidia Physiologic tests

morphology and
key morphologic
differences

Reproduction Sexual / asexual Sexual

(conjugation)/
asexual (budding)
B. CONIDIA
C. DIMORPHIC FUNGI ● Spore-like asexual reproductive structures not
● Capable of growth as a mold or yeast depending on produced by cleavage, conjugation, or free-cell
environmental conditions formation.
● Molds ● Only formed by the imperfect fungi
○ At room temperature
○ Infective to man ● Conidia morphology is important in fungal identification.
● Yeast ● Conidia classification is based on conidia morphologic
○ At 37°C development.
○ Tissue/ in vivo/ invasive ○ Microconidia
■ Single-celled, small conidia.
○ Macroconidia
■ Multicellular, large conidia

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 C. SPORES 2. ASEXUAL SPORES
1. SEXUAL SPORES
● Arthroconidia
● Zygomyces / Zygospore ○ From fragmentation of hyphal cells
○ Many sexual spores produced in large sack-like
zygospore produced in the position where 2
haploid cells have fused

● Blastoconidia
○ From budding yeast

● Deuteromyces
○ Major group of fungi
○ No known sexual stage
○ Fungi imperfecti

● Basidiomyces
○ Diploid nuclei in a club- like structure (basidium)
undergo meiosis and haploid spores bud in chains
from the tip of basidium.

● Chlamydoconidia
○ From terminal or intercalary hyphae.

● Ascomyces ● Phialoconidia
○ Single cells undergo meiosis and the resulting 4 or ○ From vase-shaped conidiogenous cells called
8 haploid spores are contained in a sac (ascus) Phialides.
○ Derived from the wall of the original diploid cell

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● Poroconidia
III. FUNGAL REPRODUCTION
○ A conidium produced through a small pore in a
conidiogenous cell.
● Sexual Reproduction
○ It involves the vegetative form of fungi or the
mycelium
○ Involves fusion of two mycelia
■ Haploid to diploid to spore formation
○ Requires the formation of specialized fungal
structures called spores.
○ Perfect fungi
■ Fungi that undergo sexual reproduction

● Annelloconidia
○ A conidium produced by an annellide.

● Asexual Reproduction
○ The mycelium will enter mitosis producing the
spores and the spores will undergo germination
○ Only involves the division of the nucleus and
cytoplasm
○ Imperfect fungi
■ Fungi that undergo asexual reproduction
■ Only fungal group to produce conidia

IV. FUNGAL MEDIA

● Sporangiospores A. SABORAUD DEXTROSE AGAR (SDA)
○ Characteristic asexual spores of the order
Mucorales. ● General-purpose
● Nutritionally poor medium mildly selective for fungi
● No longer commonly used

● Several different formulations available

○ In one formulation, the agar has an acidic pH (5.6)
that inhibits most bacteria.
○ Modified SDA (Emmons) has a neutral pH and
better supports the growth of fungi but is less
inhibitory for bacteria.

B. SABORAUD-BRAIN HEART INFUSION AGAR (SABHI)

● A nonselective medium for isolation of all fungi

● Contains dextrose, peptone, and brain heart infusion

● Can be made selective for dimorphic fungi

○ Addition of cycloheximide, chloramphenicol,
and gentamicin

C. BRAIN HEART INFUSION AGAR WITH BLOOD (BHIB)

● Used to grow most fungi

○ Especially those from sterile body sites
● Contains brain heart infusion and sheep blood

● Can be made selective for dimorphic fungi

○ Addition of cycloheximide, chloramphenicol,
and gentamicin.

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 D. SELECTIVE AGARS ○ This method prevents damage to the fungal
1. INHIBITORY MOLD AGAR (IMA) structure

● Used to grow most fungal pathogens 2. DIRECT EXAMINATION METHODS

● It is specially formulated to recover
cycloheximide-sensitive Cryptococcus
● Contains gentamicin and chloramphenicol
2. DERMATOPHYTE TEST MEDIUM (DTM)
● Used to isolate the dermatophytes
● Contains cycloheximide and gentamicin and phenol
red as a pH indicator
E. DIFFERENTIAL AGARS
1. POTATO DEXTROSE AGAR (PDA)
● Used to enhance conidia development
● Enhances pigment development of Trichophyton
rubrum
2. BIRD SEED / NIGERSEED / RAMTIL SEED / STAIB
3. CAFFEIC ACID AGAR
● Selective and differential media
● Used to grow C. neoformans
○ C. neoformans form black to brown colonies due
to the activity of phenol oxidase.
● Chloramphenicol can be added to make the media
selective
4. CORNMEAL AGAR
● With Tween 80
● Used to differentiate Candida spp.
4. OTHER AGAR
the
● Saline wet mount
○ Used to view fungal elements, such as hyphae,
conidia, and budding yeasts.
○ It has limited use and is most commonly
applicable for vaginal secretions to diagnose
vaginitis.
● Lactophenol cotton blue wet mount
○ Used to stain and preserve fungal elements in
culture isolates.
● Potassium hydroxide (KoH)
○ Used to dissolve nonfungal materials in skin,
nail, and hair samples.
● Gram stain
○ Can be used to view yeasts
● India ink (nigrosine)
○ Used to reveal capsules surrounding C.
neoformans found in cerebrospinal fluid (CSF).
○ However, due to low sensitivity, direct antigen
detection assays have generally replaced the
India ink wet mount.
● Calcofluor white stain
○ A Fluorochrome that stains chitin found in the
cell wall of fungi.
○ The stain is not absorbed by human tissue.
○ The slide is viewed using an ultraviolet light.
○ Fungi will appear white to blue to green
depending on the wavelength of light.
○ KOH can be added to clear the specimen of
cellular debris

● Agars containing rice, casein, and other nutrients are
used to differentiate Trichophyton spp.
F. CULTURE CONSIDERATIONS
● Fungal cultures are incubated at 30°C
● Growth requires from several days to several weeks
● Cultures should be maintained in a high-humidity
environment
3. HISTOLOGIC STAINING
● Gomori methenamine-silver stain
○ Fungi are dark gray to black
● Periodic acid Schiff (PAS) reaction
○ Fungi are hot pink to red
● Gridley fungus stain
○ Fungi are purplish rose with a yellow background

1. SLIDE PREPARATIONS ● Calcofluor white stain

○ Immunofluorescent stains are available for some
● Tease mount method fungal pathogens
○ A dissecting needle is used to pull apart a fungal
colony, which is placed on a slide.
○ This method may damage fungal structures,
V. MEDICALLY IMPORTANT FUNGI
especially conidia.
○ It may take several attempts to obtain a specimen A. DERMATOPHYTES
with intact conidia.

● Cellophane tape method Genera Skin Hair Nails

○ Cellophane tape is used to transfer aerial
hyphae from the colony to a microscope slide for Trichophyton X X X
examination.
Microsporum X X No infection
● Slide culture method
○ Uses a block of agar overlaid with a cover slip. Epidermophyton X No X
○ Fungal colonies are grown on the side of the agar infection
block.
○ The cover slip is removed and used for
microscopic examination.
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 Microconidia Macroconidia Colony CLINICAL FEATURES OF DERMATOPHYTE INFECTIONS
T. Grape-like Cylindric Cottony to Skin disease Location of Clinical features Fungi most
mentagrophytes Spherical Smooth-walled granular
lesions frequently
responsible
T. rubrum Small Cylindric White colony
Pear-shaped Smooth-walled with red
pigment (on Tinea corporis Non-hairy, Circular patches with Trichophyton
reverse) (ringworm) smooth skin advancing red, rubrum
vesiculated
border, and central Epidermophyton
T. tonsurans Elongate Cylindric Flat scaling. floccosum
Smooth-walled Powdery to
velvety Pruritic
With
red-brown
surface (on Tinea pedis Interdigital Acute: itching, red T. rubrum,
reverse) spaces on vesicular.
feet of E.
persons Chronic: itching, floccosum,
M. gypseum Abundant Tan wearing shoes scaling,
Thin echinulate- Powdery fissures Trichophyton
walled mentagrophytes

E. floccosum None Smooth-walled Flat Tinea cruris Groin Erythematous scaling T. rubrum,
Clavate in small Velvety lesion in
clusters Tan to olive intertriginous area. E.
green floccosum,
Pruritic
Trichophyton
B. CUTANEOUS MYCOSES mentagrophytes

Tinea unguium Nail Nails thickened or T. rubrum,

FORMS OF DERMATOPHYTOSIS BASED ON LOCATION crumbling distally,
ON THE BODY discolored; E.
lusterless. floccosum,

Dermatophytosis Features Usually associated Trichophyton

with tinea pedis mentagrophytes
Tinea pedis Involves the feet
May become
(Athlete’s foot or Alipunga) secondarily
Most prevalent of all infected with
dermatophyses bacteria

Tinea unguium Involves the nails Tinea barbae Beard hair Edematous, T. rubrum
erythematous lesion
(Onychomycosis) Trichophyton
Most difficult to treat mentagrophytes

Requires months of oral Trichophyton

verrucosum
azole or terbinafine as
well as surgical removal of Tinea capitis Scalp hair. Circular bald patches Trichophyton
nail Endothrix: with short hair stubs mentagrophytes
fungus inside or broken hair within
Tinea corporis Involves the body hair haft. hair follicles. Microsporum
Ectothrix: canis,
fungus on the Kerion rare.
Tinea cruris Involves the groin area surface of hair Trichophyton
(Jock itch or Hadhad) Microsporum infected tonsurans
hairs fluoresce.
Tinea manus Involves the hands
Dermatophytid Usually sides Pruritic vesicular to No fungi
and flexor bulbous lesions present in lesion
Tinea capitis Involves the scalp aspects of
(Buni) fingers, palm, Allergy to the
Ectothrix - formation of or any site of fungi (May
the body become
dense sheaths of spores secondarily
around the hair infected with
(Microsporum sp.) bacteria)

Endothrix - formation of
spores within the hair shaft
(T. tonsurans, T. violaceum) C. ENDEMIC/SYSTEMIC MYCOSES

Tinea barbae Involves the areas with ● Mode of Transmission

facial hair ○ Inhalation of infective stage of fungi.

● Disease: Pneumonia
● Endemic in certain geographic regions
○ Usually present as asymptomatic among
immunocompetent individuals.
○ There will be a widespread systemic presentation
in the immuno-compromised population.
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 ➢ Southwestern US
● Caused by a Dimorphic Fungi ➢ California
○ Caused by thermally dimorphic fungi
geographically restricted to specific areas of ■ Transmission
endemicity ➢ Inhalation of spores from soil, rodents

● General pathogenesis ■ Diagnostic feature

○ Inactivation of inhaled spores via alveolar ➢ Presence of spherules (filled with
macrophages endospores)
■ Initiation of antibody and cell-mediated
immunity ■ Clinical features
➢ Valley fever / San Joaquin Valley fever /
● Clinical presentation Desert rheumatism
○ Most infections are asymptomatic or mild and ★ Self-limited, influenza-like illness,
resolve without treatment malaise, cough, arthralgia
○ A small but significant number develop pulmonary
disease (pulmonary form) and may disseminate to ■ Remarks
other organs (disseminated form) ➢ Filipinos are most susceptible

● Examples: ■ Treatment
➢ Amphotericin B
○ Histoplasma capsulatum ➢ Itraconazole
■ Epidemiology ➢ Fluconazole
➢ Global distribution
➢ Mississippi ○ Paracoccidioides brasiliensis
➢ Ohio River valleys ■ Epidemiology
➢ Latin America
■ Transmission
➢ Inhalation of spores from avian and bat ■ Transmission
habitats (guano) ➢ Inhalation of spores
➢ Alkaline soil ➢ Unknown (soil)

■ Diagnostic feature ■ Diagnostic feature

➢ Presence of macrophage filled with ➢ With large, multiply budding yeasts
Histoplasma oval yeasts ★ Mariner’s wheel or captain’s wheel

■ Clinical features ■ Clinical features

➢ Pancytopenia, oral ulceration, ➢ South American blastomycosis or
splenomegaly Brazilian blastomycosis
➢ Erythema nodosum ★ Ulcerated granulomas, lytic bone
lesions
■ Treatment
➢ Amphotericin B ■ Treatment
➢ Itraconazole, ➢ Amphotericin B
➢ Fluconazole ➢ Itraconazole
➢ Fluconazole
○ Blastomyces dermatitidis
■ Epidemiology
Summary of Endemic Mycoses
➢ Eastern and Central US
➢ Great Lakes
Mycosis Etiology Tissue Form
■ Transmission Histoplasmosis Histoplasma Oval yeasts, 2 x 4 µm,
➢ Inhalation of spores capsulatum intracellular in
➢ Unknown (riverbanks) macrophages

■ Diagnostic feature Coccidioido- Coccidioides Spherules, 10 – 80 µm,

➢ With broad-based budding yeasts mycosis posadasii containing endospores, 2 –
4 µm
■ Clinical features Coccidioides
➢ Ulcerated granulomas immitis
➢ Lytic bone lesions
Blastomycosis Blastomyces Thick-walled yeasts with
dermatitidis broad-based, usually
■ Remarks single, buds, 8 – 15 µm
➢ Mimicker of squamous cell carcinoma
Paracoccidioido Paracoccidioides Large, multiply budding
■ Treatment -mycosis brasilensis yeasts, 15 – 30 µm
➢ Amphotericin B
➢ Itraconazole
➢ Fluconazole

○ Coccidioides immitis
■ Epidemiology

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 D. PHARMACOLOGY

Common Drug of Choice for Fungal Infection

Drugs Mechanism of Action

Polyenes Binds to ergosterol in fungal cell

Amphotericin B membranes, forming leaky pores

Azoles Inhibit fungal P450-dependent

Ketoconazole enzymes (lanosterol
Fluconazole 14-a-demethylase) blocking ergosterol
Itraconazole synthesis
Posaconazole
Voriconazole Resistance can occur with long-term
use

Terbinafine Inhibits epoxidation of squalene

Echincandins Inhibit B-glucan synthase decreasing

Caspofungin fungal cell wall synthesis
Micafungin
Anidulafungin

Flucytosine Blocks nucleic acid synthesis by

inhibiting DNA and RNA polymerases

Griseofulvin Interferes with microtubule function

in dermatophytes and may also inhibit
the synthesis and polymerization of
nucleic acids

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