MEDICAL MYCOLOGY

FLORDELIZA A. DE GUZMAN, RMT, MAT- PHYS

Dept. of Medical Technology
College of Nursing, CvSU
MIDYEAR 2021
 Overview of the Microbial World
• Anton van Leeuwenhoek - Dutch biologist:
- beasties in a water droplet (with his homemade
microscope)

- father of protozoology & bacteriology

• Prokaryotes – NO true nucleus nor nuclear
membrane. NO mitochondria, no endoplasmic
reticulum (ER), nor golgi apparatus
• Eukaryotes – with complex cell structures in
which the genetic material is organized into a
membrane bound nucleus
 Overview , Microbes. . . Cont

1. Bacteria – unicellular; NO nuclear membrane and

true nucleus; classified. as prokaryotes (NO
mitochondria, Endoplsmic Reticulum, nor golgi apparatus)
2. Parasites – eukaryotic as unicellular, ex.
protozoa; obtain their nutrition through
ingestion, some capable of locomotion (motile);
- locomotive structures such as flagella (whip-
like, pseudopodia (false feet); or cilia (eyelash)
- others are nonmotile;
- others are multicellular (ex. tapeworm)
 Overview , Microbes. . . Cont

3. Fungi – heterotrophic; eukaryotes, obtain nutrients

through absorption
a. Yeasts – unicellular fungi, reproduce asexually; true
yeast do NOT form hyphae or mycelia
Multicellular fungi have filaments (hyphae) which
interweave to form mats called mycelia
b. Moulds – filamentous forms, reproduce asexually and
sexually
c. Dimorphic – Some fungi assume both morphologies
(yeast and hyphae/mycelia forms), growing as yeast at
incubator or human temperature; and filamentous form
at room temperature; dimorphic fungi produce systemic
diseases in human hosts
 Overview , Microbes. . . Cont

4. Viruses – smallest infectious particles

(virions); virion – virus outside of the cell
• Cannot be seen in an ordinary light
microscope;
• Neither prokaryotic nor eukaryotic;
• Affects on cell lines (viral diseases) such as:
a) inclusions – rounding up of cells
• Syncytium – cell fusion of host cells into
multinucleated infected forms
 Overview , Microbes. . . Cont

How to distinguish cells with viral disease?

• Viruses consist of DNA or RNA, but NOT both; genome may be
double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) ,
double-stranded RNA (dsRNA), or single-stranded RNA (ssRNA).
• Are acellular (NOT composed of cells), lack cytoplasmic membranes,
and are surrounded by a protein coat.
• Are obligate intracellular parasites that require host cells for
replication (increase in number does NOT involve mitosis, meiosis,
or binary fission) and metabolism; lack enzymes, ribosomes, and
other metabolites; “take over” host cell function to produce virus;
growth (increase in size) does NOT occur in viruses.
• Viruses are mostly host and/or host cell specific:
Ex. a) human immunodeficiency virus (HIV) infects T helper
lymphocytes, NOT muscle cells, in humans
b) rabies – can infect dogs, skunks, bats, and human (brain)
• bacteriophage – virus that and possibly destroys bacterial cells
 Taxonomy

Taxonomy acc. to classification & identification

1. Genotype - genetic makeup of an organism,
or combinations of forms of one or a few
genes under scrutiny in an organisms genome.
Ex. of genotypic characteristics: - basic
sequencing of DNA or RNA and DNA base
composition ratio to measure the relatedness
of 2 organisms
2. Phenotype – readily observable physical and
functional features of an organism expressed
 Taxonomy . . . Cont.

. . . of phenotypic characteristics: a)
macroscopic (colony morphology on media)
b) microscopic (size, shape, arrangement
into groups or chains of organism morphology,
c) staining characteristics (gram-positive or
gram-negative;
d) nutritional requirements, physiologic and
biochemical characteristics
e) succeptibility or resistance to antibiotics
or chemicals
 Taxonomy. . . Cont.

Taxonomy acc. to levels of classification or subsets:

• Domain – ex. Bacteria/Archaea; Eukarya for
plants and protists
• Kingdom – animalia, protista, monera
• Phyla (division),
• Family
• Genus (genera)
• Species
• Strains
 Taxonomy, cont.

• Recent taxonomists - - organisms are in 3 domains

(replaced some kingdom). Each of these domains
are divided into kingdom based on similarities of
RNA,DNA and protein sequences:
• Prokaryotes includes domains Archaea
(archaeobacteria) & bacteria (Eubacteria)
– Archaea –not encountered in clin. Microbio; ex. are salt
loving and heat loving cells (extremes)
• Eukaryotes -includes fungi,parasites,algae, protzoa,
animals and plants; larger and contain membrane
encased organelles (w/ specific function) or
compartments
 Eukaryotes:
Cytoplasmic structures;
• nucleus – has DNA w/ discrete chromosomes (genes)
covered w/ basic proteins called histones. Nucleus has
bilayered nuclear membrane
• nucleolus – site of ribosomal RNA synthesis
• ER – membranes, as rough ER, covered with
ribosomes the site of protein synthesis; ribosomes
gives the rough appearance
- as smooth ER,NO ribosomes so does NOT synthesize
protein but synthesize phosholipids (like rough ER)
• Golgi apparatus – modify and package protein sent to
it by rough ER
 Eukaryotes. . . cont

Other membrane enclosed organelles:

• Mitochondria – main site of energy production
- contain their own DNA and the electron transport system
that produce energy for cell functions
• Lysosomes – contain hydrolytic enzymes for degradation
of macromolecules and microorganisms within the cell
• Peroxisomes – contain protective enzymes that break
down H2O2 and other peroxides generated within the cell
• Chloroplasts – for plant cells, site of photosynthesis
(energy production), produces glucose from CO2 and H2O.
Fungi are NOT plants and therefore have NO chloroplasts.
 Microbial Nutritional requirements for
Growth
1. Autotrophs –grow simply using CO2 as the sole source of
carbon with only water and inorganic salts required in
addition.
- Autotrophs obtain energy by : a. photosynthesis
(phototrophs)
b. oxidation of inorganic compounds
(chemolithotrophs)
2. Heterotrophs – require more complex substances for
growth; require an organic source of carbon such as
glucose, and obtain energy by oxidizing or fermenting
organic substances (glucose as both carbon and energy
source)
 Environmental factors affecting growth –

1. obligate aerobes – requires Oxygen for

growth(bacteria)
2. aerotolerant anaerobes – facultative aerobes,
can survive in the presence of Oxygen but not
use Oxygen in metabolism
3. Obligate anaerobes – cannot grow in the
presence of oxygen
4. Facultative anaerobe – can grow w/ or w/o
Oxygen
5. Capnophilic - organisms grow best when the
atmosphere is enrich with extra CO2 (5% to 10%)
 Host-microbe relationship:

• Symbiosis – living together ; both benefit from

one another (host and organism) - mutualism
• Commensalism –organism benefits but NO
beneficial or harmful effect to the host
• Parasitism –one specie (organism/microbe)
benefits at the expense of the other (host)
 Fungi are Everywhere

In Medicine

Food

Spoilage

Superficial Colonisation
Fungi are Eukaryotic, spore-bearing,
heterotrophic organisms that produce
extracellular enzymes and absorb their
nutrients.
Fungi – Kingdom Mycota
Divisions - Phyla – Chytridiomycotina,
Glomeromycota, Zygomycota,
Basidiomycota, Ascomycota, Microsporidia,
Neocallimastigomycota
 Mycology

Fungi are dependent upon enzyme systems to

derive energy from organic substrates.
• Saprophytes - live on dead organic matter,
enriched by nitrogenous matter, but are capable of
maintaining a separate existence as an
opportunistic pathogen in humans or animals.
Fungal infections are generally NOT
communicable in a usual sense from person-to-
person
• parasites - live on living organisms
• Dermatophytes –fungi that infect the skin and grow
in the keratin layer
 Primary Route of Infection

(Ebel, F.)
 Fungi
• Humans become accidental host for fungi by inhaling
spores or by the introduction of fungal elements into tissue
by trauma. Humans are relatively resistant to fungal
infections, EXCEPT for disease caused by dimorphic fungi.
• Alteration in the host particularly in the immune system
causes fungal infection (opportunistic fungi);
• or caused by immunosuppressive agents or serious
underlying disease - - lead to infection by organisms that
are normally nonpathogenic (ex. progressive infections as
HIV, Diabetes mellitus,
• or in patients with impaired immunologic function resulting
from corticosteroid or antimetabolite chemotherapy
(immunocompromised)
• No fungus species can be considered completely innocuous
(not harmful). Mushrooms look innocuous but are deadly.
 Mycology
Fungi are essential in recycling of elements,
especially carbon.
Life would be impossible w/o fungi - decomposers
Role of fungi in the economy:
1. Industrial uses of fungi -
• Mushrooms. (Class Basidiomycetes); 200 edible
• Truffles. (Class Ascomycetes)
• Natural food supply for wild animals.
• Yeast as food supplement, supplies vitamins.
• Penicillium roquefort - ripens cheese, adds flavor
(roquefort cheese – French cheese from goat’s and
ewe’s (female sheep) milk, contain blue mold); P.
camemberti
• Fungi used to alter texture, improve flavor of
natural and processed foods.
 Mycology
2. Fermentation
• Fruit juices (ethyl alcohol).
• Saccharomyces cerevisiae - brewer's and
baker's yeast.
• Fermentation of industrial alcohol, fats,
proteins, acids, etc.
3. Antibiotics -
• First observed by Fleming; noted
suppression of bacteria by a
contaminating fungi of a culture plate.
• Penicillium notatum – antibiotic produced
by deuteromycete
 Mycology
Role of Fungi (Cont.)
4. Plant Pathology – most plant diseases are
caused by fungi - Chestnut blight, Dutch
Elm disease, Apple scab, Brown rot
- many smuts (produce black powdery
masses of spores) and rusts attack cereals

Agaricus campestris – mushroom found in

the grocery
Shitake mushroom - -
 Role of fungi, Cont.
5. Medical importance -
• 100-150 species are recognized human
pathogens.
• Most prefer to be free-living saprophytes; and
only accidentally become pathogens.
• To be pathogenic, they must tolerate the
temperature of the host site and possess an
enzymatic system that allows them to utilize
animal tissues
• Increased incidence of fungal infections in recent
times
• Some fungi cause human diseases:
- ringworm, athlete’s foot, Histoplasmosis (systemic
disease)
 Mycology
• Cyclosporine – drug used to suppress immune
responses in patient’s receiving organ transplant
was produced from fungi
• Some fungi are poisonous ( cyclopeptides are
toxic substance found in mushrooms)
• Ergot – type of fungus (Claviceps purpurea);
causes nervous spasm, convulsion, psychotic
declusion, gangrene
• In 1722, Ergot was called St. Anthony’s fires and
its outbreak caused many to be burned at the
stake as witches
 Mycology
Importance of Medical Mycology
• During the time period between 1941 - 1973, the
number of reported deaths in the U.S. due to
scarlet fever, typhoid, whooping cough,
diphtheria, dysentery and malaria decreased
from 10,165 cases to 107; but reported deaths
due to mycoses during the same time period,
increased from 324 to 530.
• Increased mobility - We can travel to a
geographical area where a fungus exists as part
of the comensal flora of the local population, or
is endemic to the area.
• We have an aging population.
 Mycology
The Immunosuppressed Patient:
• AIDS
• Drugs - immunosuppressant drugs
used in organ transplant patients,
cancer and leukemia patients.
 Mycology
Immunology of the Mycoses
Antibody mediated immunity (B-cell, humoral)
• Humoral immunity- immnune response by B
lymphos w/c produce circulating antibodies
(immunoglobulins) in reaction to infectious
organisms and other foreign antigens
• Antibodies are often produced in response to a
fungal infection, but do not confer immunity.
• Serological tests for identification of fungal
diseases detect these antibodies.
Cellular mediated immunity (T-cell)
• T-cell immunity is effective in resistance to fungal
infections.
Fungi
•Of the 50-250,000 fungal species less than 200 cause
human disease and approx. 25 species cause the majority
of human disease
•Yeasts: unicellular fungi reproduce by budding
• Moulds (filamentous): produce hyphae and mycelium
• Dimorphic: grow as moulds (environment) or yeasts (in
human host)

Yeast
Hyphae
 Definition of fungi (summary)

1) Eukaryotic cells - contain membrane bound cell organelles

including nuclei, mitochondria, golgi apparatus,
endoplasmic reticulum, lysosomes etc; non-vascular
organisms. Eukaryotes also exhibit mitosis. These features
separate fungi from bacterial which are prokaryotic cells
lacking the above structures.
2) Heterotrophic - fungi lack chlorophyll and are therefore
not autotrophic (photosynthetic) like plants and algae;
rather they are heterotrophic absorptive organisms that are
either saprophytes (living on dead organic matter) or
parasites (utilizing living tissue).
3) Like plants, fungi have rigid cell walls - therefore non-
motile, a feature which separates them from animals. But
few have a motile phase (ex. Chytrids)
Basic Structure

Woronin body

Hyphal Tip
 Structure of Fungi.
Fungi occur in two basic growth forms
(morphology) or stages:
(a) yeast form - morphologically, as a single-celled
fungus unicellular, 2 to 60 μm; reproduced by
simple budding to form blastoconidia. On culture,
Colonies are usually moist or mucoid (or smooth
& creamy), opaque
Ex. Cryptococcus neoformans (capsulated yeast)
Yeast-like fungi may be basidiomycetes, or
ascomycetes such as Candida albicans; grow
partly as yeast w/ partly elongated cells
resembling hyphae (called pseudohyphae)
 Dimorphic fungi
• occur in 2 forms: a) yeast b) molds
• Mostly caused systemic infections (host –
tissue):
a) Histoplasma capsulatum
b) Blastomyces dermatidis
c) Paracoccidiodes brasiliensis
d) Coccidiodes immitis
e) Pennicillium marneffei
f) Sporothrix schenkii (sub cutaneous)
 Dimorphic

• Several systemic fungal pathogens

exhibit either a yeast ( or yeastlike)
phase and filamentous forms
• Thermally dimorphic – when
dimorphism is temperature dependent:
- produce mold form at 25oC to 30oC
- produce yeast form at 35oC to 37oC
Cont. Structure of fungi

b) filamentous or mold form – colonies are

fluffy, cottony, wooly, or powdery; a vegetative
and aerial growth of filaments (hyphae); form
true mycella. Structures such as mushrooms
consist simply of a number of filaments packed
tightly together, and reproduce different types
of spores or conidia. Moulds produce a great
variety of conidia which are borne on specialized
hyphae or conidiophores. Many moulds can be
identified by the morphology of these spores
and by their arrangement on the hyphae. Ex.
Rhizopus, Mucor
Cont. structure. .

• Fungal filaments are known as hyphae and a

mass of hyphae collectively make up the
mycelium. The terms "hyphae" and "mycelium"
are used interchangeably. There are two kinds of
hyphae; non-septate (coenocytic) and
(aseptate)

septate. The septa divide the hyphae into

compartments but not into cells. In some groups
nuclei and/or cytoplasm can flow through a hole
or pore in the centre of these septa.
• non-septate – has continuous cells w/ many
nuclei
Cont. structure

• Fungi with non-septate hyphae typically belong to the

Zygomycetes. Non-septate hyphae are
considered to be more primitive because if a hyphal
strand is damaged the entire strand dies
• Septate hyphae are typically found in the
Basidiomycetes and Ascomycetes, the latter also
includes the hyphomycetes or conidial fungi. When a
septate hyphal strand is damaged, the pores between
adjacent compartments can be plugged, thus
preventing death of the whole hyphal strand. (woronin
body crystal)
• Vegetative structures involved growth and catabolism
 The Fungal Cell Wall
Rigid structure surrounding the cell
Cell wall
•The fungal cell wall is essential for growth and viability
(development); contains chitins (rigidity & support),
mannans, polysaccharides
•Pathogen associated molecular patterns (PAMPS) – the
immune systems of most organisms recognize fungal cell
wall components such as ß-d-glucan and mannans
•Melanin – is an important component of fungal cells walls
especially in spores. It protects against UV radiation and
Reactive Oxygen Species
•Composition can affect action of antimicrobial agents –
Candida mutants lacking mannosylphosphate in their cell
wall displayed enhanced resistance to cationic antimicrobial
peptides via reduced peptide binding (Harris et al. 2009)
Immune Avoidance
•Cryptococcus neoformans – produces a
polysaccharide capsule – possible functions in
avoiding phagocytosis, affects antibodies – capsule
deficient mutants are less virulent (Perfect, 2005)
•Hydrophobin layer of Aspergillus conidia renders
them inert to the immune system (Aimanianda et
al., 2009)
•Cytoplasm – has ergosterols
•Has true nuclei, paired chromosomes – divide
sexually, asexually or both
 Reproduction in fungi

• Sexual - formation of Zygospore, ascospores or

basidiospores
• Ascomycetes• Sexual spores called ascospores are
present within a sac like structure called Ascus.
• Several asci may be seen within a fruiting body as seen in
Penicillium, Aspergillus• Each ascus has 4 to 8 ascospores.
• Asexual reproduction – budding or fission
• Asexual spores are formed on or in specialized
structures. Vary in size, shape & color but these
characteristics are constant for a particular species
• Asexual spores - Sporangiospores: present within a
swollen sac- like structure called Sporangium
• Micro conidia - Small, single celled
• Macro conidia – Large, single or many celled
 Fungi Need to Eat

Heterotrophic
Secrete extracellular enzymes
Absorptive nutrition
(Osmotrophs)

•Saprobes: decay dead organic

matter
•Pathogens: biotroph, necrotroph
•Symbionts: parasites -
commensals - mutualists
 Fungi Interact with other organisms
•Fungi may gain nutrients
through parasitism of other
organisms

Plant Parasitism

“The Frog problem”

Chytridiomycosis
Affecting 30% of the
amphibian species of
the world??

Nematode Parasitism
Mycotoxicosis
 Mycotoxins
• Mycotoxins are low-molecular-weight
secondary metabolites of fungi
• Often produced by food spoilage organisms or
in basidiocarps (Mushrooms)
• mycotoxins are important chronic dietary risk
factor
• Aflatoxins - Aspergillus spp.(from peanuts);
• Citrinin – Penicillium spp.;
• Ergot Alkaloids – Claviceps spp. – Ergotism;
Fuminosins – Fusarium spp.
 Amanita muscaria
http://www.doctorfungus.org

• Genus/Species: Amanita muscaria • Slide Reference #: GK 016

• Image Type: Macroscopic Morphology • Disease(s): Mycotoxicosis

(Basidiocarps)
 Amanita phalloides
http://www.doctorfungus.org

• Genus/Species: Amanita phalloides • Slide Reference #: GK 017

• Image Type: Macroscopic Morphology • Disease(s): Mycotoxicosis

(Basidiocarp)
 Reproduction
• Fungi can reproduce asexually (imperfect) or sexually (perfect)
• Asexual reproduction – results in the formation of conidia
following mitosis
- conidiogenous cells – fruiting structure forming conidia that
contain genetic material necessary to create a new fungal colony:
a. phialide – vaselike producing phialoconidia
b. annelide – ringed producing annelloconidia
- both form their conidia blastically (budding) like many yeasts
c. arhtroconidia – formed by fragmentation of fertile hyphae
• Sexual reproduction – joining of 2 compatible nuclei followed by
meiosis
- teleomorph – term for fungus that reproduce sexually
- occasionally, a teleomorph may reproduce asexually w/c is
called anamorph
- synanamorph – if more than one anamorph, Ex.
Pseudallescheria boydii
Fungal Spores
 Mycology
Sporulation & Spores (continued)
• Asexual spores - most common type.
– Conidia - asexual fungal spores borne
externally in various ways from a
conidiophore;(develop at a hyphal tip) often referred to
a macroconidia (multicellular) and
microconidia (unicellular).
– Arthroconidium (Arthrospore) - special type
of asexual spore formed by disarticulation of
the mycelium. (hyphal fragmentation)
– Blastoconidia (Blastospore) - asexual spore
formed from a budding process along the
mycelium or from another blastospore.
 Mycology
• Asexual Spores (continued)
– Chlamydospore - thick-walled asexual
spore formed by direct differentiation of
the mycelium (concentration of
protoplasm and nutrients).
– Sporangiospore - an asexual spore
contained in a sporangium (within a sac) at the
end of a sporangoiphore (develop in a sac).
– Thallospore - asexual spore produced on
a thallus (hypha).
 Hyphae

• Septate

• Aseptate
 Hyphae

• Hyaline

• Dematiaceous
 Mycelium
• Mass of branching intertwined hyphae
– Vegetative
– Aerial
– Fertile
 Two Types of Structures Associated with
Sexual Reproduction
1. Sporangia – produce spores
spores – non-motile cells produced on aerial hyphae;
spread by wind or animals
sporocarp – a large reproductive structure that
produce spores; the familiar part of the mushroom
2. Gametangia – produce gametes
zygote – cell formed by 2 gametes
fungal cells contain haploid nuclei
haploid – a # of chromosomes in the normal gamete,
equal to half the # in the normal somatic cell
Two basic types of reproductive propagules
are found in the fungi:
1) Sexual propagules - produced by the fusion of two
nuclei that then generally undergo meiosis.
• Sexual methods of reproduction involve plasmogamy
(cytoplasmic fusion of two cells), karyogamy (fusion of
two nuclei), genetic recombination and meiosis. The
resulting haploid spore is said to be a sexual spore, e.g.
zygospores, ascospores and basidiospores. If a sexual
spore is produced only by fusion of a nucleus of one
mating type with a nucleus of another mating type (+
and - strains), the fungus is said to be heterothallic. In
contrast, homothallic moulds produce sexual spores
following the fusion of two nuclei from the same strain.
 Aspergillus (Anamorph)
Mitosporic State

Neosartorya (Teleomorph)
Sexual State
 Sexual Reproduction

• Ascospores
– Ascus
– Ascocarp

Basidiospores

Zygospores
 Mycology
Sporulation & Spores - preferred terms when
there is a merging of nuclear material. Self-
fertile are termed homothallic. Mating types are
termed heterothallic.
• Sexual spores - exhibit fusion of nuclei.
– Ascospore - spore formed in a sac-like cell
known as an ascus. Often eight (8) spores
formed; developed during sexual
reproduction (Ascomycetes)
– Basidiospore - sexual spore produced on a
specialized club-shaped structure, called a
basidium. (Basidiomycetes)
– Zygospore - a thick-walled spore formed
during sexual reproduction (Phycomycetes)
 Cont.Types of reproductive propagules

2) Asexual propagules - either spores or conidia

depending on their mode of production, and
which arise following mitosis of a parent nucleus.
Conidia arise either by budding off conidiogenous
hyphae or by differentiation of preformed
hyphae. Asexual spores are commonly formed by
consecutive cleavages of a sporangium. Asexual
forms of reproduction represent the major
method for the maintenance and dissemination
of many fungi.
 Mycology
Sporulation & Spores (continued)
• Asexual spores - most common type.
– Conidia - asexual fungal spores borne externally in
various ways from a conidiophore; often referred to a
macroconidia (multicellular) and microconidia
(unicellular).
– Arthroconidium (Arthrospore) - special type of
asexual spore formed by disarticulation of the
mycelium.
– Blastoconidia (Blastospore) - asexual spore formed
from a budding process along the mycelium or from
another blastospore.
 Mycology
• Asexual Spores (continued)
– Chlamydospore - thick-walled asexual
spore formed by direct differentiation
of the mycelium (concentration of
protoplasm and nutrients).
– Sporangiospore - an asexual spore
contained in a sporangium at the end
of a sporangoiphore.
– Thallospore - asexual spore produced
on a thallus (hypha).
 Asexual Reproduction
• Conidia
• Conidiophore

• Arthroconidia
 Asexual Reproduction
• Blastoconidia

• Pseudohyphae

• Chlamydoconidia
• Chlamydospores
 Asexual Reproduction
• Macroconidia
• Microconidia

• Phialoconidia
• Phialide
 Asexual Reproduction
• Annelloconidia
• Annellide

• Sporangiospores
• Sporangium
• Sporangiophore
 Mycology
Miscellanous terms:
• Ascus - sac-like structure containing (usually eight)
ascospores developed during sexual reproduction
in the Ascomycetes.
• Conidiophore - a specialized branch of hypha on
which conidia are developed.
• Dematiaceous - pigmented, dark in color, usually
gray to black.
• Hyaline - colorless, clear.
• Dermatophyte - fungus that causes superficial
mycoses.
 Mycology
Miscellanous terms: (continued)
• Diphasic (dimorphic) - the ability of some fungi to
grow as either yeast or filamentous stages,
depending on conditions.
• 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.
• Germ Tube - small projections which arise from
cells of certain yeasts; indicates the onset of
hyphal formation.
 Mycology

Miscellanous terms: (continued)

• Pseudohyphae - a chain of elongated budding
cells that have failed to detach (not true
hyphae).
• Rhizoids - root-like structures.
• Sporangiophore - a special aerial hypha or stalk
bearing a sporangium.
• Sporangium - a sac or cell containing spores
produced asexually.
• Sterigmata - a specialized structure that arises
from a basidium and supports basidiospores.
 Mycology
Classifications of Fungi:
1) Geographic grouping - where they exist.
2) Epidemiologic grouping - how organism is
transmitted.
3) Taxonomy grouping - according to
morphologic and culture characteristics.
4) Topographic Grouping - type of mycosis
produced.
 3 Epidemiological Groups:
1. Geophilic – in contact with soil
2. Zoophilic – in contact with animal
host
3. Anthropophilic – in contact with
man
 Mycology
Topographic Grouping of Fungi: (most often used)
1) Superficial - Confined to the outermost layers of
the skin and hair. No host cellular or inflammatory
response due to organisms being remote from
living tissue. Essentially no pathology; the disease
is recognized purely on cosmetic basis.
2) Cutaneous - in the keratin of the skin, nails, and
hair. These organisms prefer non-living cornified
layers. The disease is called a dermatophytosis or
dermatomycosis. Host response is patchy scaling
or eczema eruptions. They are classified according
to the area of the body that is involved.
 Mycology
Topographic Grouping of Fungi: (continued)
3) Subcutaneous - Involve the deeper layers
of skin and often muscle tissue. Man is an
accidental host following inoculation of
fungal spores via some form of trauma. This
type of infection is often identified by the
presence of a characteristic tissue reaction
or granule.
4) Systemic - Attack the deep tissues and
organ systems; often creating symptoms that
resemble other diseases.
 Mycology
Categories of systemic disease:
• Those caused by truly pathogenic fungi with the
ability to cause disease in the normal human
host when the inoculum is of sufficient size
(Histoplasma capsulatum, Blastomyces
dermatitidis, Coccidioides immitis,
Paracoccidioides braziliensis).
• Those caused by opportunistic fungi, low
virulence organisms, which require the patient's
defenses to be lowered before the infection is
established (Aspergillus spp. Candida albicans,
Cryptococcus neoformans).
 Tinea/Pityriasis versicolor
Malassezia furfur
normal skin commensal, lipolipic yeast
Tinea nigra - only on palms and soles of the feet,
black or brown
Black piedra   White piedra  
Dermatophytosis (Epidermophyton floccosum)
Tinea capitis Tinea barbea
Tinea corporis Tinea cruris (jock itch)
Tinea manuum Tinea pedis
 Tinea unguium
thickening & discoloration of the nail
(onchomycosis)
Ectotrix – hair loss endotrix - hair breakage
Chromoblastomycosis
Mycetoma
 Mycology
Saprophytes:
• Contaminates or opportunistic pathogens?
• Most are inhibited by cycloheximide.
• Grouped by type of mycelia produced.
– Septate vs. aseptate
– Hyaline vs. dematiaceous
Mycology

Saprophytes:
Hyaline Members -
Aspergillus spp.
• Growth rate varies, colors vary,
surface velvety to cottony.
• Mycelium - septate and hyaline with
unbranched condiophores
(compare to Syncephalastrum,
which appears similar, but is
aseptate).
• A. fumigatus is considered a
potential pathogen, especially if
from a pulmonary source.
Aspergillus Structure
Aspergillus fumigatus

Blue/Green colony
Phialids with spores are
Directed upward
Aspergillus flavus

Green colony
Orange colored spores
that surround the vesicle
Aspergillus niger

Black colony
Black spores surround the vesicle
 Aspergillus terreus

Sandy colored colony

Resistance to Amphotericin B
Mycology

Saprophytes:
Hyaline Members -
Paecilomyces spp.
• Rapid grower, colors vary.
• Brush-like conidiophores.
• Long, tapered sterigmata.
Mycology

Saprophytes:
Hyaline Members -
Penicillium spp.
• Commonly rapid growing;
white to bluish-green.
• Conidiophores
characteristically form a
brush-shaped structure.
• Sterigmata are flask shaped.
Penicillium species – most common mold
in the environment, bread mold, uncommon
cause of human disease
Mycology

Saprophytes:
Hyaline Members -
Scopulariopsis spp.
• Moderately slow growing.
White turning brown with age.
• Branched or unbranched
conidiophores.
• Sterigmata are coarsely
roughened.
Mycology

Saprophytes:
Hyaline Members -
Trichoderma spp.
• Moderately rapid growth.
• Flask-shaped conidiophores.
• Conidia are clustered.
Mycology

Saprophytes:
Hyaline Members -
Fusarium spp.
• Rapid growth, white colonies may
be come brightly colored.
• Short conidiophores often
branched, have macro- and micro-
conidia, which are oval to sickle-
shaped.
• Has been reported to cause eye
infections.
 Mycology

Saprophytes:
Dematiaceous Members -
Alternaria spp.
• Rapid growth; colonies become
very dark with age; may
become overgrown with looser
white to gray aerial mycelium.
• Conidiophores bear single or
branched chains of large,
brown conidia.
Mycology

Saprophytes:
Dematiaceous Members -
Curvalaria spp.
• Rapid growth. Velvety
colonies vary in color from
grayish-brown to black.
• Spirally arranged brown
conidia are borne at the tips.
• Brown, septate, unbranched
conidiophores.
Mycology

Saprophytes:
Dematiaceous Members -
Cladosporium spp.
• Rapid growth. Green
colonies, reverse is black.
• Septate, dematiaceous
mycelium.
• Conidia are borne in
chains.
 Mycology

Saprophytes:
Aseptate Members -
• All are susceptible to cycloheximide.
• Rapid growers.
• Some have root-like structures termed
rhizoids.
• Spore bearing structures are called
sporangiophores.
Mycology

Saprophytes:
Aseptate Members -
Mucor spp.
• Very rapid growth; can fill a
culture tube in one day.
• Unbranched sporangiophores.
• No rhizoids
Rhinocerebral
mucormycosis
Mycology

Saprophytes:
Aseptate Members -
Rhizopus spp.
• Rhizoids are present.
• Sporangiophores nodal in
origin.
Mycology

Saprophytes:
Aseptate Members -
Syncephalastrum spp.
• Very rapid growth. White to dark
gray colonies with dense, cottony,
aerial mycelium.
• Aseptate, hyaline mycelium, with
short, branched sporangiophores,
terminating into tips.
• Many tubular sporangia containing
chains of spores.
Mycology
Yeasts:
Candidia albicans
• Cutaneous infections such as
oral thrush or vaginitis, but
can become systemic.
• ID by positive germ tube test,
or production of
chlamydospore on cornmeal
agar.
 Candida albicans
• Intertrigo is a rash that usually affects the
folds of the skin, where the skin rubs together
or where it is often moist. This rubbing can
cause a breakdown in the top layers of the
skin, causing inflammation and a rash. The
breakdown of skin makes it easier for bacteria
or fungus to develop in this area.15 Apr 2020
Candidiasis
culture at 37 degrees C
 Candida albicans
Identification

C. glabrata only forms yeast

No pseudohyphae
 Mycology

Yeasts:
Cryptococcus neoformans
• Associated with pigeon feces.
• Has thick capsule.
• Grows at 37 degrees & produces
melanin-like pigment on caffeic
acid agar.
• ID in CSF by India ink stain for
capsule.
India ink
Mycology

Yeasts:
Geotrichum spp.
• Commonly present in GI
tract.
• Implicated in respiratory
infections; frequently as a
secondary invader.
• Causes disease in the
immunosuppressed.
• Produces arthrospores.
Mycology

Bacteria - Like Fungi:

• Some produce sulfur granules
(Actinomyces israeli)
• Often require special media,
stains, & conditions for
growth, i.e. anaerobic
(Actinomycetes spp).
• Some are partially acid-fast
(Nocardia spp.)
Mycology

Dimorphic Fungi:
Sporothrix schenkii
• Rose fever, gardeners
often affected.
• Infection via traumatic
implantation.
Mycology

Dimorphic Fungi:
Coccidioides immitis
• San Joaquin valley fever.
• Mycelial phase present in
culture.
• Yeast phase in tissue.
 Mycology

Dimorphic Fungi:
Histoplasma capsulatum
• Mississippi Valley fever.
• Associated with bird
H. capsulatum mold phase droppings
• Disease mimics tuberculosis.
• Found frequently in
reticuloendothelial cells as
extracellular inclusions.
H. capsulatum yeast phase
Appearance in culture at 30 degrees C
Histoplasma capsulatum
culture at 30˚ C is white and
cottony.

Microscopic exam:
Tuberculate [projections]
macroconidia is the
structure used for ID.
Microconidia are the
infectious particle.
Appearance in culture at above 35
degrees C (Hist. cap.)
 Mycology

Dimorphic Fungi:
Blastomyces dermatitis
• North American
Blastomycosis (Gilcrest
disease).
• Large yeast cells with
single bud.
Blastomyces culture at 37
degrees C
Slow growing yeast colony taking @ 4
weeks to form a colony
Yeast cell is 8 – 20 um in size and is
unique for it’s Broad Based Budding
pattern and the double contoured wall.
Gridley stain from skin lesion,
reveals the central round
mother cell with series of
narrow- necked buds that
look like spokes of wheel
 Mycology

Dimorphic Fungi:
Paracoccidioides braziliensis
• South American
Blastomycosis.
• Large yeast cells with
multiple buds (mariner’s
wheel).
 Mycology

Superficial mycoses:
Malassezia furfur
• Cause of pityriasis versicolor
(tinea versicolor).

Phaeoannellomyces werneckii
• Cause of superficial
phaeohyphomycosis (tinea
nigra).
Tinea nigra grow only on palms and soles of the feet
(brown or black patches) 
Black piedra   White piedra  
 Mycology
Superficial mycoses:
Trichosporon beigelii (now T. asahii)
• Cause of white piedra.
• Commonly affects pubic hair, axillary hair, beards,
eyebrows
• May be T. ovoides for scalp hair
Piedraia hortae (Horta, 1911)
• Cause of black piedra.
• Affects scalp hair

• Piedra (Spanish for stone)

 Mycology

Cutaneous mycoses:
Microsporum spp. - in hair, skin, rarely nails;
frequently in children, rarely in adults;
often spontaneous remission occurs
(ringworm).
Trichophyton spp. - hair, skin & nails; in both
children & adults (athlete’s foot; T. rubrum).
Or ringworm or tinea pedis
Epidermophyton spp. - skin, nails, rarely hair;
in adults, rarely in children (ringworm).
 Mycology

Cutaneous mycoses:
Microsporum spp.
• Macroconidia are attached singly.
• Thick walled.
• Mature forms are echinulate
(spiny).
Mycology

Cutaneous mycoses:
Trichophyton spp.
• Macroconidia are attached
singly.
• Have smooth walls.
 Mycology

Cutaneous mycoses:
Epidermophyton spp.
• Macroconidia are attached in
multiples.
• Smooth walls (beaver tails).
• Grows slowly.
 Mycology
Subcutaneous mycoses:
Mycetoma:
• Eumycotic Mycetoma -
– Clinical picture similar to actinomycotic mycetomas.
Disease is slowly progressive, with patients
frequently dying of secondary infections.
– Treatment consists of excising early, localized
lesions (amputation may be required for advanced
infections). Anti-fungal drugs have little effect.
– Body tries to wall off the offending fungus. Invasion
of deeper tissue may follow, with bone involvement,
draining sinus tracts, or progression through the
lymphatics.
Mycetoma
 Mycology
Subcutaneous mycoses:
Mycetoma:
• Localized, tumorous lesions in cutaneous and
subcutaneous tissues usually the foot.
Nodules are formed, and a collection of pus
and formation of sinuses results.
• Actinomycotic mycetomas must be
differentiated from Eumycotic (true fungi)
mycetomas and as they have greatly differing
treatments.
 Mycology
Subcutaneous mycoses:
Mycetoma:
• Actinomycotic mycetoma -
– “Lumpy-jaw” - The initial nodules formed are
firm and described as "woody" or "lumpy.“
– Sulfur granules - Bread crumb-like aggregates
of microorganisms and cellular debris which
take on the appearance of a sulfur particle.
– Caused by Nocardia spp. & Streptomyces spp.
– Advanced cases often require amputation due
to physical impairment.
 Mycology
Subcutaneous mycoses:
Chronic, supperative or granulomatous infections of the
subcutaneous tissues, usually on an extremity (hands,
feet); can extend through the lymphatics or form sinus
tracts. Caused by a variety of fungi and bacteria-like
fungi that live in the soil.
Chromoblastomycosis:
• Non-contagious skin diseases characterized by the
development of a warty lesion that has a cauliflower
appearance. Occurrence is usually on the legs or feet.
Caused by Fonsecaea pedrosoi, Cladosporium spp.,
and Phialophora spp. Fonsecaea pedrosoi is the most
common etiologic agent of chromoblastomycosis
worldwide.
Chromoblastomycosis
 Mycology
Systemic mycoses:
• Histoplasma capsulatum -
– Histoplasmosis - an infection of the
reticuloendothelial system resulting in patchy
bronchopneumonia containing yeast-laden
phagocytic cells within alveolar spaces.
– Worldwide in distribution, it is endemic in the
Mississippi, Missouri, St Lawrence, and Ohio
river valleys.
– Strong association with bird and bat
droppings.
 Mycology
Systemic mycoses:
• Coccidioides immitis -
– Coccidioidomycosis - usually an asymptomatic or
mildly symptomatic self-limiting upper respiratory
tract infection, but may become disseminated and
fatal.
– San Joaquin Fever, or Valley Fever - it is endemic in
southwestern US, esp. Arizona and California.
– Caution must be taken with cultures; organism
produces arthroconidia which are very easily
inhaled.
 Mycology
Systemic mycoses:
• Paracoccidioides brasiliensis -
– Paracoccidioidomycosis - also called
South American blastomycosis.
• Blastomyces dermatitidis -
– Blastomycosis - also called North
American blastomycosis or Gilcrest
disease.
 Safety Issues (Myco lab)

1. Use of class 2 biological safety cabinet – due to

hazard of airborne conidia; Specimen processing
and plating must be performed in it
2. Use of enclosed incinerators to eliminate the
hazards of gas flames and to contain particles
emitted when loops or needles are incinerated
3. Cabinet should be checked daily to see that none
of the airflow inlets or outlets is blocked by
supplies, incinerators, or waste disposal jars
4. Use of petri dishes is hazardous; screw-top tubes
is recommended
 Petri dish vs. screw capped tubes

• Petri dish – hazardous • Screw-top tubes –

release of airborne release of airborne
conidia conidia is less
• Screw –capped tubes –
• Have greater surface less media dehydration
area for colony isolation than petri dishes
and are easier to - more easily handled
manipulate when
preparing for
microscopic
examination
 Specimen Collection, Handling, Transport

1. All specimen for mycology should be

transported and processed as soon as possible
ASAP because many pathogenic fungi grow
slowly
2. Any delay in processing compromises specimen
quality and decreases the probability of isolating
the causative agent due to overgrowth of
contaminants
3. All laboratories should reject the unsatisfactory
or improperly labeled specimens (protocol)
 Specimen Collection, handling,Transport
4. Specimen should neither be frozen nor allowed
to dry; should be left at RT
5. All work should be done in biosafety cabinets
6. Swabs are generally rejected for fungal culture
7. For skin scrapings: wipe w/ alcohol before
collection
-Skin scrapings should be collected from outer
edge of lesions (edge of glass slide)
- put scrapings in paper or envelope to prevent
blowing by the wind
Specimen Collection, handling , Transport
8. Hair (including root) should be pulled with
sterile forceps
9. Nail specimens: disinfected ROH (70%
isopropyl alcohol); submitted as scrapings,
cuttings or as complete nail
• Almost any tissue or body fluid can be submitted for
fungal culture
• The most common specimens:
1. respiratory secretions 5. tissue
2. hair 6. blood
3. skin 7. bone marrow
4. nails 8. CSF
hair, skin, or nails submitted for dermatophyte
culture are generally contaminated with bacteria
rapidly or rapidly growing fungi or both – thus
primary isolation should contain antimicrobial
agents
Pls. see p. 630, identification of fungal isolates
 Laboratory Methods in Medical Mycology
Collection, Handling and Processing of Specimens
• Skin - cleaned with 70% isopropyl alcohol to remove
dirt, oil and surface saprophytes; scraped; KOH wet
mount
• Nails - cleaned same as for skin, 70% isopropyl
alcohol . Usually clipped (cut); need to be finely
minced before inoculating to media
• 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 –
• Body fluids - normal sterile collection procedures
 Collection, Handling and Processing of Specimens

• Blood and bone marrow

• Blood from septicemic patients can harbor
both known pathogenic and opportunistic
fungi ; lysis centrifugation to recover fungi
-use biphasic system (broth and agar)
• Heparinized bone marrow – plated directly
onto media at bedside
• Blood culture bottles NOT recommended
• Blood/bone marrow - generally inoculated
directly to BHI broth and BHI slant.
 Laboratory Methods in Medical Mycology
Preparation of Specimens for Transport to Laboratory
• Hair & nails sent in a dry envelope, inside proper
container
• Other specimens are usually sent frozen or on dry ice
• Packaging - biohazard regulations. Any growing
cultures must be on tube media (not plates). Aluminum
screw-capped inner tube with outer cardboard mailing
tube
• Inside labeling information: Patient ID, specimen
source, suspected organism
• Outside labeling information - must state:
WARNING: POTENTIAL PATHOGEN
 Laboratory Methods in Medical Mycology
Appropriate Processing of Specimen to Recover
Fungus
• Skin, nails, & hair - direct exam following KOH
preparation
• CSF – centrifuged to be concentrated before
inoculation; examine sediment microscopically,
inoculate media; no need of antimicrobial agents
because CSF is normally sterile
• Pleural fluid, sputum, and bronchial aspiration -
specimen must be fresh as saprophytes would
overgrow pathogens such as H. capsulatum.
Specimens may be refrigerated up to 2 hours
• Gastric washings - same as for pleural
fluids
• Genito-urinary specimens - first
morning specimen preferred ;
centrifuged & sediments used to
inoculate media
• Abscess Fluid, Wound
exudates,Tissues – use of dissecting
microscope for the presence of
granules
 Laboratory Methods in Medical Mycology
Appropriate processing of specimen to recover
fungus (continued)
• Wound abscess or drainage - should be
cultured anaerobically, especially if
actinomycosis is suspected
• Tissue specimens - examine for pus,
caseous material or granules; mince
aseptically, can use small amount of sterile
saline and the supernatant inoculated
 Microscopic Examination, Stains . .
1. KOH prep –used for clearing/dissolving of
cellular debris; wet mount; direct
–heat increases rate of clearing; can
detect yeasts, hyphae and spore;
–10%: for skin and hair; 20-40%: for
nails
2. Calcofluor White – fluorescent dye binding
to chitin; increased sensitivity
– (+):yellow-green fluorescence
 Microscopic Examination

• KOH w/ Calcofluor White – 1 gtt of

calcofluor white (a fluorescent dye)
added to KOH preparation before adding
a cover slip. Calcofluor binds to
polysaccharide present in the chitin of
the fungus or to cellulose.
• Fungal elements fluoresce apple green or
blue-white
 Microscopic. . .

3. India ink –negative stain; detects encapsulated

yeast (Cryptococcus neoformans) in CSF
4. Gram stain (Hucker’s modification) –all fungi
are Gram (+); detects yeast
- Gram Stain - most fungi are gram positive;
Actinomyces and Nocardia are gram variable
5. Giemsa/Wright stain –primarily detects H.
capsulatum in blood/BM
 Micros . . . Stains
6. Lactophenol cotton blue (Aman’s medium)
–used for fungal cultures NOT direct --
Lactophenol Cotton Blue (LPCB) - very
popular for quick evaluation of fungal
structures; will stain the chitin in cell
walls of fungi
7. Wood’s Lamp –uses UV light to detect
naturally fluorescing fungi (for Tinea capitis) –
glow w/ greenish or bluish color)
 Micros. . . Stains
8. Tissue stains: PAS, H&E(Hematoxylin & Eosin),
Grocott-Gomori (GMS); Masson-Fontana;
Seller’s
• - Periodic Acid - Schiff Stain (PAS) - stains
certain 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

• Staining is done when KOH test is negative

 Stains Used in Mycology

• 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
• Modified Acid-Fast Stain - differentiates
acid-fast Nocardia from other aerobic
Actinomyces
 GENERAL STEPS IN IDENTIFICATION

 1. Direct microscopy (KOH Mount/Calcofluor White)

2. Culture isolation (2 weeks for dermatophytes; 3-4
weeks for systemic fungi)
3. Gross examination of culture (Morphology,
Changes in media)
4.LPCB Staining
5. Microscopy (of isolates): usually definitive
6. Biochemical Testing (VITEK)/CHO Assimilation
test: usually not helpful
7. Serology (antigen detection)/PCR (nucleic acid
amplification)
 Laboratory Methods in Medical Mycology
Direct Examination of Specimens
• Direct microscopic exam is required on any material
sent to lab for fungus culture. Look for spores,
hyphae, mycelial elements, budding yeast, mycotic
granules
• Wet mount prep - good for yeast; examination is
done in natural environment, so loss of fragile
structure is minimized
• KOH prep - done on skin scrapings, hair, nails,
sputum, vaginal specimens, etc. The KOH clears
the specimen’s tissue cells, mucous, etc., so fungal
elements can be seen
 Laboratory Methods in Medical Mycology
Fungal Culturing
• Tubed media is used rather than plated media because:
– there is less chance for spore release into the environment
– less chance for dehydration
– ease of storage
• The agar in a tube is inoculated in a straight line.
• The agar on plates is Inoculated like a large "S" so that rapid
growing fungi can be removed
• Incubation should be aerobic (and anaerobic if Actinomycetes
are suspected)
• Incubate cultures at room temperature & also at 37oC if
dimorphic fungi are suspected
• Cultures are kept for 4 weeks & should be examined every
other day
• Systemic pathogens often require 10 days to 2 weeks
 CULTURE MEDIA
1. Saboraud’s dextrose agar (SDA)
–general/nonselective media; pH 5.6
2. SDA with antibiotics (Mycosel/Mycobiotic) –contains
cycloheximide and chloramphenicol; detects
dermatophytes and most of the pathogenic fungi –
Cycloheximide  an antifungal antibiotic produced by Streptomyces
griseus, an organism which also produces streptomycin (Whiffen,
1948).
3. Dermatophyte test medium (DTM) – uses phenol red
as indicator
4. Brain heart infusion agar (BHIA) – for dimorphic fungi
 
 Culture media, cont.

5. Potato dextrose agar (PDA) –induces

sporulation and pigmentation
6. Cornmeal Tween 80 agar –induces
chlamydospores for C. albicans
7. Cornmeal agar w/ 1% glucose –
Trycophyton rubrum produces red
pigment
8. Staib’s niger seed/bird seed agar –
isolation of C. neoformans
 Culture Media, cont. .
9. Czapek’s medium –isolation of Aspergillus
spp.
10. Rice medium –distinguishes
Microsporum canis (+) from M. audouinii
(-)
11. Urea agar –differentiates C. neoformans
(+) from Candida and other yeasts (-)
–also differentiates Trycophyton
mentagrophytes (+) from T. rubrum (-)
12. Cotton seed agar –for B. dermatitidis
 Laboratory Methods in Medical Mycology
Media Used for Isolation of Fungi
• Sabouraud's dextrose agar (Sab-Dex) - classic medium,
recommended for most studies
• 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)
• 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
 Laboratory Methods in Medical Mycology
Special Applications 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
• KT Medium & Kelley Agar - used to convert dimorphic fungus
Blastomyces dermatidis from mycelial to yeast form
• Modified Converse Liquid Medium (Levine's) - used to
promote spherule production by Coccidioides immitis
• Christensen's urea agar - urea is hydrolyzed by some yeast
to form ammonia. The pH increases turning the media from
yellow to dark pink
 Incubation

• 30oC -fungi
• 37oC – dimorphic fungi
• 4-6 weeks, examined twice weekly for growth
• Zygomycetes such as Mucor and Rhizopus
grow rapidly and may fill the tube w/ aerial
mycellium w/in few days
 Laboratory Methods in Medical Mycology
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.
Color may be on fungus itself, on its sporulating apparatus,
on the agar, or on the bottom of the colony (reverse
pigmentation). Pigment color is due to the color of the
sporulating apparatus. Pigment can diffuse into the agar. It is
important to note the top pigment (obverse); the underside
pigment (reverse); and any discoloration of the medium
 Laboratory Methods in Medical Mycology

Microscopic Evaluation of Fungi

• Teased Preparations – part of the colony is
removed to a slide with inoculating needle.
Lactophenol cotton blue (LPCB) 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; tape placed
onto a drop of LPCB on a slide & examined
 Laboratory Methods in Medical Mycology
Biochemical Studies Used to ID Yeast and Yeast-like
Organisms
• Carbohydrate assimilation & fermentation( instead of Sugar)
– 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
• Nitrogen assimilation (KNO3)
– Utilizes 3 tubes with different sources of nitrogen.
Bromthymol blue is the indicator (blue to yellow is
positive).
 Laboratory Methods in Medical Mycology

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
ANTIMICROBIAL SUSCEPTIBILITY TESTING
1. Fungicidal Agents
a. Amphotericin B, Nystatin –targets cell
membrane (ergo sterol)
b. Azole (Fluconazole) –also targets cell membrane
c. Griseofulvin –inhibits mitosis
2. Methods –all detects MIC (no established
susceptibility patterns)
a. Broth microdilution method
b. E-test method –well-defined MIC; easy to handle
c. Colorimetric
 Laboratory Methods in Medical Mycology
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
 4 Divisions (Phyla)of Fungi -
common
1. Zygomycota
2. Ascomycota
3. Basidiomycota
4. Deuteromycota
 ZYGOMYCETES (bread or pin moulds)

• Fast growing, terrestrial, largely saprobic fungi with non

motile cells; cosmopolitan (665 species).
• Hyphae coenocytic and mostly aseptate. Asexual spores
include chlamydoconidia, conidia and sporangiospores
contained in sporangia borne on simple or branched
sporangiophores. Sexual reproduction is isogamous
producing a thick-walled sexual resting spore called a
zygospore. However,
most isolates are heterothallic i.e. zygospores are absent,
therefore identification is based primarily on
sporangial morphology. Medically important orders and
genera include:
 ZYGOMYCETES
• Zygomycota includes black bread mold and
molds, such as those of the genus Glomus, 
that form important symbiotic relationships
with plants. Most are soil-living saprobes that
feed on dead animal or plant remains. Some
are parasitic of plants or insects. They
reproduce sexually and form tough zygospores
from the fusion of neighboring gametangia.
There is no distinguishable male or female.
 Ascomycete
• Ascomycota includes yeasts, the powdery
mildews, the black and blue-green molds, edible
types such as the morel and the truffle, and
species that cause such diseases of plants as
Dutch elm disease, chestnut blight, apple scab,
and ergot. There are over 50,000 species, about
25,000 of which occur only in lichens. In
ascomycetes, the hyphae are subdivided by
porous walls through which the cytoplasm and
the nuclei can pass. Their life cycle is a complex
combination of sexual and asexual reproduction.
 Basidiomycete – the club fungi
• Basidiomycota includes the gill fungi (most 
mushrooms), the pore fungi (e.g., the bracket fungi,
which grow shelflike on trees, and an edible type
called tuckahoe), and the puffballs. It also includes
the fungi that cause smut and rustin plants. Like
ascomycetes, the hyphae are subdivided by porous
walls. In basidiomycetes, two hyphae fuse to form a
dikaryotic mycelium (a mycelium in which both
nuclei remain distinct). These mycelia differentiate
into reproductive structures called basidia that
make up the basidiocarp (the body popularly
known as the mushroom cap).
 Basidiomycete. . cont
• The nuclei then fuse and undergo meiosis,
creating spores with one nucleus each. When
these spores germinate, they produce hyphae,
and the process begins again.
• Agaricus campestris – mushroom found in the
grocery
 Deuteromycete
• Deuteromycota comprises a miscellaneous
assortment of fungi that do not not fit neatly in other
divisions; they have in common an apparent lack of
sexual reproductive features. Also called Fungi
Imperfecti, the group includes species that help create
Roquefort and Camembert cheeses, that cause
diseases of plants and of animals (e.g., athlete's foot
and ringworm), and that produce penicillin. A number
of the fungi classified as deuteromycetes have been
found to be asexual stages of species in other groups,
and some classification schemes consider the
deuteromycetes a class under Ascomycota.
 Common Mold-Allergic Responses

1. Allergic Rhinitis - affects 20-30% of the

population. Other than molds, dust mites and
mite fecal pellets, pollen or other plant
fragments, and animal dander such as from
pets or mice can cause or contribute to this
complaint.
 Common Mold-Allergic Responses
2. Hypersensitivity pneumonitis, or extrinsic
allergic alveolitis, may be caused by airborne
biological particles. Some experts, (Arora, A.S.,
"Understanding the Health Effects of Mold," vi-
vii, Synergist magazine, September 2003) assert
that this is a rare problem today but we suspect
that it may be a risk where water-cooled air
conditioning systems or mishandled air
conditioning condensate are used or present.
Common Mold-Allergic Responses
3. Asthma - affects about 5% of the U.S.
population. Several molds commonly found
indoors may be a factor in triggering an asthma
attack, including the most common
genera, Cladosporium sp. and Alternaria sp.
Others, Penicillium sp. and Aspergillus sp. which
we often find indoors in buildings with a history
of water or moisture problem are asthma
triggers. Various other factors besides mold can
cause or contribute to asthma attacks.
Examples of Mold Related
Illnesses
 Examples of Mold Related Illnesses

1. Aspergillosis - affects people who have become

sensitized to Aspergillus sp., an inflammation of the
respiratory airways. Aspergillosis should not be
considered an allergy and is potentially fatal.
2. Blastomycosis a potentially fatal fungal infection
(localized lung infection or a skin infection) caused by
inhaling Blastomyces dermatitidis, primarily affects
farmers, gardeners, potentially also infections by the
fungus entering a cut in the skin. (Diagnosed by
examination of bodily fluids, assisted by x-ray for lung
assessment), lung infections can be successfully
treated with proper medication. Some sources
describe alternative treatments for skin infections.
 Examples of Mold Related Illnesses

3. Cryptococcus infection (cryptococcosis): (from bird droppings),

at primary risk are people with compromised immune system
but normal adults can also be infected by inhalation
of Cryptococcus neoformans, leading to a form of meningial
encephalitis. 

Cryptococcosis is a fungal disease caused by Cryptococcus

neoformans or Cryptococcus gattii. Most people do not get sick
with cryptococcosis, but some people are more likely than
others to get this disease. For these people, cryptococcosis can
cause serious symptoms of lung, brain and spinal cord disease,
such as headaches, fever, cough, shortness of breath, and night
sweats. Cryptococcus neoformans is found in bird
droppings. Cryptococcus gattii is found in soil around trees.
 Examples of Mold Related Illnesses

4. Histoplasmosis (from bat droppings). This is the most

commonly occurring infection in this group, caused
by inhalation of Histoplasma capsulatum. In people
with compromised immune system this can be a
particularly dangerous infection, potentially fatal if
not treated. See also granuloma, iritis, lung nodules.
5. Tinea pedis (from skin contact). Athlete's foot, also
called tinea pedis, is the most common type of fungal
infection. It's closely related to other fungal infections
such as ringworm and jock itch. Although contagious,
athlete's foot often can be treated with over-the-
counter antifungal medications.
• https://www.ncbi.nlm.nih.gov>books
• Anaerobic condition – generator is activated
with water. Oxygen within the jar and the
hydrogen that is generated are converted to
water in the presence of the catalyst. CO2 is also
generated, required by some anaerobes
• Capnophilic – candle inside the jar
• pH – fungi prefer acidic condition
• Nutritional immunity – sequestration of
essential micronutrients like Fe
Fungal culture, Capnophilic, extra CO2
 Candida albicans

• Causes diseases of the skin – (intertrigo) and

nails (onychia & paronychia)
• paronychia– inflammation of the folds of the skin
bordering the nail beds
• Involve mucous membranes thrush of mouth,
vagina, & anus
• thrush – white eruptions
• Cause systemic disease of the lungs or
endocardium or military septicemic spread
• Although potential pathogen is a normal
inhabitant of GIT
 Candida albicans

• Direct exam from skin, mucous membrane,

sputum,
- ovoid bodies w/ small buds
• Saboraud’s medium, 37oC or at room temp -
wine odor, yeast like colonies w/ budding
• Corn meal agar
• sugar fermentation –
• Pathogenic to lab animals – 1% saline
suspension of organism given w/in 7 days
Intravenously, rabbit – will kill it
 Contaminant fungi that rarely cause disease

1. Aspergllus
2. Penicilium
3. Geotrichum
4. Mucor
Watch:
• How to plate a fungal culture on a petri
dish YouTube · Haviks4 Jan 2017 (1.53 min)
• Slide Culture Technique for Fungi - Amrita University
YouTube · Amrita Vlab28 May 2011
3 types of media
 REFERENCES

• https://www.medicalnewstoday.com/articles/tinea-nigra
• https://emedicine.medscape.com/article/1092330-overvi
ew

• https://www.medicalnewstoday.com/articles/322331
• https://www.healthline.com/health/skin-lesion-koh-exam
• https://www.google.com/search?q=what+is+cycloheximi
de+used+for&oq=what+is++
cyclohexamide&aqs=chrome.2.69i57j0i10l2j0i390.20070j
0j15&sourceid=chrome&ie=UTF-8
• https://www.diffen.com/difference/Mold_vs_Yeast
• https://www.britannica.com › science › Reproductive-pr.
• https://familydoctor.org › condition › intertrigo
• http://www.life-worldwide.org › fungal-diseases › fungal-Fungal