Mycology

Is the study of the

fungi
 Fungi
50,000 + valid species
and fewer than 100
have been identified
as human pathogens
Classified as thallophytes
Eukaryotes
Heterothrophic members
Fungi stain gram ____,
and require oxygen to
survive
Role of fungi in the economy:
 Mycology
Antibiotics
First observed by
_______; noted
suppression of
bacteria by a
contaminating
fungus of a culture
plate.
 Comparison of fungi and bacteria
Feature Fungi Bacteria
diameter 4 m 1 m
nucleus eukaryotic prokaryotic
cytoplasm with mitochondria and without mitochondria and
endoplasmic reticulum endoplasmic reticulum
Cell membrane sterol present sterol absent
Cell wall chitin peptidoglycan
spores sexual and asexual spore for survival, not for
spore for proliferation proliferation
dimorphism yes No
metabolism Require organic May do not require
carbon and no organic carbon and maybe
anaerobes anaerobes

6
 2 phases
1. Mold phase
Multicellular
Cottony mycelial mass
 The mycelium is
an intertwining
structure of
tubular filaments
called the hyphae
(the microscopic
unit of fungi)

Septate hyphae-
contain cross walls
Aseptate hyphae-
continous
 2 phases

2. Yeast phase
Creamy, resembling
bacterial colony
 Dimorphic Fungi
Fungi with both yeast phase at 35-37 C and mold
phase growing at 25 C
 Fungi may
reproduce
either
sexually or
asexually
Sexual reproduction-
requires the formation of
special structures so that
fertilization or nuclear
fission can occur
Fungi that exhibit the sexual
phase are known as
PERFECT FUNGI

1. Basidiospores- which
contained in club-shaped
basidium
-
-Ascospores- contained in sac-like ascus
 zygospores- involve the fusion of 2
identical cells arising from same hyphae
-oospores - involve fusion of cells from 2
separate non identical hyphae
 Asexual reproduction involves on mitosis with
nuclear and cytoplasmic division
Imperfect fungi or fungi imperfecti

Conidiogenesis conidia formation can occur

blastically or thanically
 Blastic conidiogenesis the parent cell enlarges,
septum forms, and the enlarged portion splits
off to form daughter cell
Thallic conidiogenesis the septum forms
first, and the new growth beyond the septum
becomes the daughter cell.
 Asexual spores -
Conidia - asexual fungal spores borne externally in
various ways from a conidiophore
Conidiophores may branch into secondary
segments to form phialides
(multicellular) and microconidia (unicellular)
 Arthroconidium (Arthrospore) involve the simple
fragmentation of the mycelium of the septum into cylinder
shaped or cask-shaped spores
Spores are thick walled maybe adjacent or alternate in
arrangement.
Disjunctor cells appear between arthrospores
 Blastoconidia
(Blastospore) - asexual
spore formed from a
budding process along
the mycelium or from
another blastospore.
 Chlamydospore - thick-walled, resistant
resting spores produced by rounding up and
enlargement of the terminal hyphal cells
 Sporangiospore
- an asexual
spore contained
in a sporangium
at the end of a
sporangoiphore
.
 Descriptions of common
stuctures and terms
Miscellanous terms:
Cleistothecium large round
multicellular structure the
surrounds the asci and
ascospores until a structure
ruptures, releasing the
ascospores
Dematiaceous - pigmented,
dark in color, usually gray to
black.
Hyaline - colorless, clear
Dermatophyte - fungus that
causes superficial mycoses.
Miscellanous terms:
(continued)
Ectothrixic - ability of
the fungus to grow on
the outside of a hair
shaft.
Endothrixic - ability of
the fungus to grow
and penetrate into the
hair shaft.
Miscellanous terms:
(continued)
Germ Tube - small
projections which arise from
cells of certain yeasts;
indicates the onset of hyphal
formation.
Miscellanous terms:
(continued)
Pseudohyphae - a
chain of elongated
budding cells that
have failed to detach
(not true hyphae).
Miscellanous terms:
(continued)
Rhizoids - root-like
structures.
Sporangiophore - a
special aerial hypha or
stalk bearing a
sporangium.
Sporangium - a sac or cell
containing spores
produced asexually
 Collection and Handling of
Specimens
Skin - cleaned with 70%
alcohol. Scrape outer edge of
ring in cases of suspected ring
worm. KOH exam
Nails - cleaned same as for
skin. Usually clipped; need to
be finely minced before
inoculating to media
 Collection and Handling of
Specimens

Hair - obtained from edge of

infected area of scalp. Use a
Wood's lamp (fluorescence) to
help locate infected hair. Hair
can be obtained by plucking,
brushing, or with a sticky tape
 Collection and Handling of
Specimens
Blood- Wrights or giemsa stain bottles are
used frequently
Bone Marrow- aspirate 0.5 ml marrow with
sterile syringe in heparin
Mucocutaneous- collect with scraping plaque
with tongue depressor . Transport in sterile
saline
 Collection and Handling of
Specimens
Subcutaneous tissue : lesions and abscess
Perform biopsy or needle aspiration. Tissue must
be minced or ground

Sputum, bronchial washings, tracheal aspirates

sputum should be collected during early morning
on 3 consecutive days. Avoid 24 hour collection to
avoid contamination and overgrowth.
 Collection and Handling of
Specimens
Throat
Collect in 2 sterile swabs. Scrape of and collect
material with tongue depressor
Urine
- collect clean catch midstream of first morning
void into sterile container.
Vaginal, cervical
- collect two swabs and place in transport media.
Examine microscopically
Identification methods
Saline mount
simple, quick
method to observe
fungal elements
including budding
yeast, hyphae and
pseudohyphae
- A specimen is
added to one drop
of sterile saline
- Identification is
difficult due to the
lack of contrast
 Laboratory Methods in Medical Mycology

Direct Examination of
Specimens
KOH prep - done on skin
scrapings, hail, nails, sputum,
vaginal specimens, etc. The
KOH clears the specimens
tissue cells, mucous, etc., so
fungal elements can be seen

Celluflour, a brightening agent

can be added to the koh
solution. It binds with chitin and
provides excellent contrast
 Laboratory Methods in Medical Mycology

Stains Used in Mycology

Lactophenol Cotton Blue
(LPCB) - very popular for
quick evaluation of fungal
structures; will stain the
chitin in cell walls of fungi
and will impart a blue color
to the cell walls
 Laboratory Methods in Medical Mycology

Stains Used in Mycology

Periodic Acid - Schiff Stain (PAS) - stains the hyphae of

the molds and also some yeast. PAS exidizes the
hydroxyl in the carbohydrates tocertain polysaccharide in
the cell walls of fungi. Fungi stain pink-red with blue
nuclei
 Gomori Methenamine Silver Stain - outlines
fungi in black due to the silver precipitating on
the fungi cell wall. Internal structures are deep
rose to black; background is light green

Gridley Stain - Hyphae and yeast stain dark blue

or rose. Tissues stain deep blue and
background is yellow

Mayer Mucicarmine Stain - will stain capsules of

Cryptococcus neoformans deep rose
 Fluorescent Antibody Stain - simple, sensitive, and specific. Applications
for many different fungal organisms

Papanicolaou Stain - good for initial differentiation of dimorphic fungi.

Works well on sputum smears

Gram Stain - most fungi are gram positive; Actinomyces and Nocardia
are gram variable
 Modified Acid-Fast Stain - differentiates acid-fast N____from other
aerobic Actinomyces

Giemsa Stain use on blood and bone marrow specimens.

India Ink - demonstrates the capsule of Cryptococcus neoformans in CSF
specimens
Fungal Culture
Incubation should be aerobic (and anaerobic if
Actinomycetes are suspected)
Incubate cultures at room temperature & also at 37C if
dimorphic fungi are suspected
Cultures are kept for 2-4 weeks & should be examined
every other day
Systemic pathogens often require 10 days to 2 weeks
(H.capsulatum)
 Media Used for Isolation of Fungi
Sabouraud's dextrose agar (Sab-
Dex) - classic medium,
recommended for most studies.
Contains peptone and glucose
Sabouraud's dextrose agar with
chloramphenicol -
chloramphenicol inhibits bacterial
growth
Mycosel agar - commercially
available. Contains
chloramphenicol to inhibit
bacterial growth; cycloheximide
to inhibit saprophytic fungi and
some yeasts (including C.
neoformans)
 Media Used for Isolation of Fungi
Brain heart infusion slant (BHI) - more
enriched than Sab-Dex. Used to
recover H. capsulatum

Potato-dextrose agar (PDA) and Corn-

meal agar - used in slide cultures;
these induce spore formation, which
greatly aids in identification .
 DIFFERENTIAL TEST MEDIA

Caffeic Acid Agar - Cryptococcus neoformans

will produce melanin resulting in black
colonies (protect media from light during
incubation)
Birdseed Agar - used to isolate Cryptococcus
neoformans from contaminated cultures. It
produces phenol oxydase, breaking down the
medium and resulting in production of
melanin
 DIFFERENTIAL TEST MEDIA

Cotton seed agar- conversion of mold phase

of Blastomyces dermatitidis to yeast phase
KT Medium & Kelley Agar - used to convert
dimorphic fungus Blastomyces dermatitidis
from mycelial to yeast form
Modified Converse Liquid Medium (Levine's) -
used to promote spherule production by
Coccidioides immitis
 DIFFERENTIAL TEST MEDIA

Christensen's urea agar - urea is hydrolyzed by

some yeast to form ammonia. The pH increases
turning the media from yellow to dark pink
Rice medium- Identification of Microsporum
audouinii
Yeast fermentation broth identification of yeast by
fermentation reactions with various carbohydrates
 DIFFERENTIAL TEST MEDIA
Tricophyton agars- nutritional requirement test for differentiation
of Tricophyton. Seven media contain growth factor requirement
for trycophyton species
No. 1- casein agar base
No. 2 - casein agar base + inositol
No.3 - casein agar base + inositol + thiamine
NO. 4 - casein agar base + thiamine
No. 5- casein agar base + nicotinic acid
No. 6 ammonium nitrate agar base
No. 7 - ammonium nitrate agar base + histidine
 Colony Morphology (macroscopic features)
Surface topography - some fungal colonies may
cover the entire surface of agar; others grow in a
more restricted manner
Surface texture examples: cottony or wooly
(floccose), granular, chalky, velvety, powdery, silky,
glabrous (smooth, creamy), waxy
Pigmentation - fungi may be colorless or brightly
colored.
 Microscopic Evaluation of Fungi
Teased Preparations part of the
colony is removed to a slide with
inoculating needle. Lactophenol cotton
blue stain often used
Slide Culture Techniques - provides a
view of undisturbed microscopic
morphology.
Transparent Tape Preparation clear
cellophane tape used to lift parts of a
colony to a microscope slide
 Biochemical Studies Used to ID Yeast and Yeast-
like Organisms
Carbohydrate assimilation & fermentation
Measures utilization of a carbohydrate under
anaerobic conditions as determined by acid and
gas production
Specimen is inoculated beneath broth.
Bromcresol purple is the indicator. Acid
production turns purple broth to yellow. Gas is
detected by appearance of bubbles trapped in a
fermentation tube. Observe every 48 hours for 14
days
 Biochemical Studies Used to ID Yeast and Yeast-
like Organisms
Nitrogen assimilation
Utilizes 3 tubes with different sources of nitrogen.
Bromthymol blue is the indicator (blue to yellow is
positive).
 Other Tests of Importance
Germ tube - Candidia albicans & Candidia
stellatoidea produce germ tubes when
incubated in a protein medium
Demonstration of chlamydospores - yeast is
inoculated by jabbing the appropriate agar
(Cornmeal agar with tween 80). Cultures are
observed every 24 hours for 3 days looking
for chlamydospore production
Hypersensitivity: Skin Tests and Serological Tests
Skin tests - demonstrate T-cell immunity (cellular) to
a fungus
Serological tests - demonstrate B-cell (humoral)
immunity to a fungus (acute & convalescent
specimens are required)
Complement fixation
Agglutination tests
Precipitin tests
Immunofluorescence
Immunodiffusion techniques
Counterimmunoelectrophoresis
In general, humans have a high level of innate immunity to fungi
and most of the fungal infections are mild and self-limiting.

For the exogenous pathogenic fungi, the infections are classified

according to where the infections take place:
A. Superficial mycoses
B. Cutaneous mycoses
C. Subcutaneous mycoses
D. Systemic mycoses

54
A. Superficial mycoses:
Infections are limited to the hair or the outermost layers of skin

55
56
The organism is lyophilic
and grows in areas
where sebum and skin
oil accumulates.
Involves the skin chest ,
back and upper arms
57
58
A tropical infection
involves the palms and
hand Tinea Nigra
Palmaris

SDA- black, yeasty

colonies with an olive
green mycelium
59
60
Cream colored wrinkled
colonies are seen on the
SDA in 5 days when
incubated at room
temperature

Hyaline hyphae,
blastoconidia and
arthrospores when grown
on cornmeal-Tween 80
agar
61
62
Green-black,
heaped colonies
are observed on
SDA

63
B. Cutaneous mycoses:
Fungi invade the keratinized layers of skin,
hair and nails and the diseases are limited in
these layers

The fungi causing these diseases are

termed dermatophytes

The diseases are referred to as tinea

64
Tinea may be associated with :
Antrophilic fungi infects humans
Zoophilic- which are parasitic on animals other than
humans
Geophilic free living soil saprophytes

Tinea infections are frequently seen in many animals,

including dogs, cats, which serve as a route of infection
for children

65
All tinea are caused by members of three
genera:
Trichophyton (hair,skin & nails)
Epidermophyton (skin and nails)
Microsprum (hair and skin)
Unlike the superficial mycoses, cellular
immune responses may be evoked
66
common cutaneous mycoses:

67
Some Clinical Features of Dermatophyte Infection.

Skin Disease Location of Lesions Clinical Features Fungi Most Frequently

Responsible

Tinea corporis Nonhairy, smooth skin. Circular patches with T rubrum, E floccosum
(ringworm) advancing red,
vesiculated border and
central scaling. Pruritic.

Tinea pedis1 (athlete's Interdigital spaces on Acute: itching, red T rubrum, T

foot) feet of persons wearing vesicular. Chronic: mentagrophytes, E
shoes. itching, scaling, floccosum
fissures.

Tinea cruris (jock itch) Groin. Erythematous scaling T rubrum, T

lesion in intertriginous mentagrophytes, E
area. Pruritic. floccosum
Tinea capitis Scalp hair. Endothrix: Circular bald patches T mentagrophytes, M
fungus inside hair shaft. with short hair stubs or canis
Ectothrix: fungus on broken hair within hair
surface of hair. follicles. Kerion rare.
Microsporum-infected
hairs fluoresce.

Tinea barbae Beard hair. Edematous, T mentagrophytes

erythematous lesion.

Tinea unguium Nail. Nails thickened or T rubrum, T

(onychomycosis) crumbling distally; mentagrophytes, E
discolored; lusterless. floccosum
Usually associated with
tinea pedis.

Dermatophytid (id Usually sides and flexor Pruritic vesicular to No fungi present in
reaction) aspects of fingers. bullous lesions. Most lesion. May become
Palm. Any site on body. commonly associated secondarily infected
with tinea pedis. with bacteria.
Microsporum audouinii
A slow-growing anthropomorphic
dermatophyte
Tinea capitis and Tinea corporis

Colonies of M. audouinii appear

cottony white and generally form
little salmon pink or orange
brown pigment on the reverse
Microsporum audouinii

Microscopic appearance
Bizarre shaped macroconidia,
thick walled club or spindle
shaped and multiseptate
Rare microconidia
Microsporum canis

Macroconidia are spindle-

shaped, with
echinulate, thick walls and have
3 to 15 cells

The tapering, sometimes

elongated, spiny distal ends of
the macroconidia are key
features that distinguish this
species

Microconidia are abundantly

formed
Microsporum canis

Colonies are fluffy and white that

develops bright yellow periphery,
with the reverse side of the
colony usually developing a
lemon yellow pigment
Microsporum gypseum
Numerous thick walled
cigar shaped
multiseptate
macroconidia with spiny
surfaces and rounded
tips
Sparse clavate smooth
walled microconidia

Abundant macroconidia
and microconidia
produced
Microsporum gypseum
Flat tan brown of
cinnamon brown
granular powdery colony.
Reverse side is rose
brown, red brown or
cinnamon
Epidermophyton floccosum

produces only one size of

conidia- macroconidia

The smooth, thin-walled

macroconidia are produced
in clusters or singly.
Occasionally, conidia may be
single-celled, but usually
they are separated into two
to five cells by perpendicular
cross walls
Epidermophyton
floccosum

Colonies of E.
floccosum are
yellow to yellow-
tan, flat with
feathered edges,
and remain small
in diameter
Trichophyton mentagrophytes

makes microconidia and

macroconidia.

Microconidia are primarily

globose but may appear tear-
shaped and found primarily in
clusters described as grapelike.

Macroconidia are thin-walled,

smooth, and cigar-shaped,
with four to five cells separated by
parallel cross walls.
Trichophyton
mentagrophytes

Colonial types may

be fluffy, velvety or
granular flat
White or tan to
pink. Reverse side
is white, rose red
and red-brown
Trichophyton rubrum

known to produce three- to

eight-celled cylindric
macroconidia
Tear shaped microconidia
born laterally from the long
strands of hyphae,
Pencil shaped microconidia
Trichophyton
rubrum

Fluffy or granular
white to pink
colonies. Reverse
side is cherry red
or wine red when
grown on corn
meal
Trichophyton tonsurans
leading cause of tinea
capitis in children in
many parts of the
world

Trichophyton tonsurans
possess microconidia
that are extremely
variable in shape, tear,
club or balloon shaped
microconidia
Trichophyton tonsurans

Flat powdery , granular

and cream tan yellow
rose or rust colored
colony. Colonies
become heaped or
sunken with folds.
Reverse is yellow to tan.