NAME : BARAT, JEHANI J.

DATE: JUNE 16, 2022

COURSE: BSMT 2A

LESSON 1: INTRODUCTION TO MYCOLOGY

MYCOLOGY
: Refers to the study of FUNGI, which are EUKARYOTIC organisms that evolved in tandem
with the animal kingdom.
CHARACTERISTICS OF FUNGI
: Most fungi are NON-MOTILE________, possess a rigid cell wall (with NUCLEUS inside)
and are NON PHOTOSYNTHETIC
: Each fungal cell has at least ONE NUCLEUS with a nuclear membrane, endoplasmic
reticulum, mitochondria and secretory apparatus.
: Most fungi are obligate or facultative AEROBES
: CHEMOTROPHIC , secretes enzymes that degrade a wide variety of organic substrates
into soluble nutrients that are then passively absorbed or taken into the cell by active
TRANSPORT
DIFFERENCE OF FUNGAL CELL WITH BACTERIA AND PLANT CELLS

1. Fungal cells have RIGID CELL WALL external to the cytoplasmic membrane.
2. Cytoplasmic membrane is made up of STEROL ( ERGOSTEROL). In mammalian
cells the predominant sterol is CHOLESTEROL.
3. Fungi are usually HAPLOID in their DNA content, diploid nuclei are formed through
nuclear FUSION in the process of SEXUAL reproduction
4. Fungi cell wall does not contain PEPTIDOGLYCAN, glycerol, TEICHOIC ACID or
lipopolysaccharide. Instead, they contain complex polysaccharides such as
MANNANS, GLUCANS and CHITINS as well as structural proteins.

GENERAL FEATURES AND CHARACTERISTICS OF FUNGI

FUNGI THAT CAUSE HUMAN INFECTIONS ARE DIVIDED BASED ON THEIR
MORPHOLOGICAL FORM
1. YEAST
:SINGLE CELLS, usually SPHERICAL to ELLIPSOID in shape and varying in diameter from
3-15 MICROMETER
: unicellular; creamy resembling BACTERIAL COLONY; fungal cell

2. MOLDS
: Grows as FILAMENTOUS, tube-like structures called HYPHAE that vary in diameter from
2-10 MICROMETER
: multicellular; with cottonyMYCELIAL mass
filamentous -branching hyphae- elongated tubes
Mycelial - a hyphae that joins together
YEAST VS MOLDS

CHARACTERISTICS YEAST MOLDS

MOSTLY UNICELLULAR MULTICELLULAR WITH

STRUCTURE AND EXISTING TUBULAR FILAMENTOUS
INDIVIDUALLY OR BUDS
GROWING
ROUND/ OVAL SHAPED THREADLIKE
APPEARANCE

BUDDING OR BINARY PRODUCTION OF SEXUAL

METHOD OF FISSION OR ASEXUA
REPORDUCTION

TYPES OF HYPHAE

1. VEGETATIVE HYPHAE : Penetrates the media and absorbs food

2. Aerial hyphae: Directed ABOVE THE SURFACE OF THE MEDIA
3. Reproductive hyphae: AERIAL STRUCTURES that carries reproductive SPORES

Conidia or spores – very strong wind carried away allow the transfer of
reproductive spores
DIFFERENT STRUCTURAL APPEARANCE OF HYPHAE
 1. SEPTATE - with CROSS WALLS / septa (septated hyphae)

 2. aseptate – without WITHOUT CROSS WALLS

 3. hyaline – TRANSPARENT hyphae

 4. DEMATIACEOUS – pigmented hyphae

• when many hyphae are joined together, they form the so-called MYECELIUM
 Mycelium has 2 parts:

 a) THALLUS – VEGETATIVE part; absorbs water and nutrients

 b) aerial – reproductive part contains the fruiting bodies that produce the
reproductive structures known as CONIDIA/ SPORES
VARIABLE FUNGI
: Fungi that can TRANSITION between yeast – like and hyphal morphologies
: Variation of shape is directly related to PATHOGENESIS since different forms may be
better suited for DIFFERENT MICROENVIRONMENTS

DIFFERENT VARIABLE FUNGAL FORMS

FUNGAL FORM GROWTH PHASE

ONE GROWTH PHASE (YEAST/ MOLD)
MONOMORPHIC FUNGI

MOLD AT ROOM TEMPERATURE (20-25

DIMORPHIC FUNGI DEGREE CELSIUS) NEED ROOM ONLY/
CABINET
YEAST AT BODY TEMPERATURE (37
DEGREE CELSIUS) NEEDED INCUBATOR
MOLD AT ROOM TEMEPRATURE
DIPHASIC FUNGI YEAST AT 27 DEGREE CELSIUS IN
TISSUES

METHODS OF REPRODUCTION
Fungi may reproduce either ASEXUAL or sexual REPRODUCTION
1. Anamorph
: The ASEXUAL FORM form and its reproductive elements are termed CONIDIA.
: Involves MITOTIC division of the HAPLOID nucleus and is associated with
production by BUDDING, spore-like CONIDIA_ or by the separation of HYPHAL ELEMENTS

2. Teleomorph
: The SEXUAL form and its reproductive elements are called SPORES (Ascospores,
zygospores and basidiospores)
: Involves the HAPLOID nuclei of donor and recipient cells FUSE to form a diploid
nucleus, which then divides by classis MEIOSIS.
SPORES INVOLVED IN ASEXUAL REPRODUCTION (ANAMORPH)

SPORES DESCRIPTION IMAGE

Spores produced single or
Conidia multiply in long chains or clusters
by specialized vegetative hyphae
known as CONIDIOPHORES

MACRONIDIA: Large,
Multicellular

Microconidia: SMALL,
UNICELLULAR

Develops as DAUGHTER cell

BLASTOCONIDIA buds off the MOTHER and is
pinched off
(Blastospores)

Thick walled, resistant, resting

spores produced by rounding up
Chlamydoconidia and ENLARGEMENT of hyphal
(Chlamydospores part
)
TERMINAL: End of hypha
Intercalary: WITHIN HYPHA
SESSILE: Side of hypha

Involves the simple

Arthroconidia fragmentation of the MYCELIUM.
(Arthrospores)
It appears “JOINTED”,
RECTANGULAR / BARREL
shaped spores
Sporangiospores Spores contained in a
SPORANGIA or sacs that are
produced TERMINALLY on a
aseptate hyphae
(SPORANGIOPHORE)

SPORES INVOLVED IN SEXUAL REPRODUCTION (TELEOMORPH)

SPORES DESCRIPTION IMAGE

ASCOSPORES Spores are

contained in a
saclike ASCUS

Involves the fusion of

ZYGOSPORES two IDENTICAL
CELLS arising from
the same HYPHAE.

Involves the fusion of

Oospores cells from two
SEPARATE, NON
IDENTICAL hyphae

BASIDIOSPORES Spores are

contained in a club –
shaped BASIDIUM
MODE OF TRANSMISSION
Usually acquired from the external environment via the ff
1. INHALATION of infectious CONIDIA generated from environmental molds (some are
UBIQUITIOUS and some are restricted by climate that favors their growth)
2. IMMUNOCOMPROMISED patients
3. TISSUE penetration and invasion

SPECIMEN COLLECTION
1. Skin specimens should be cleaned with 70% ALCOHOL to remove dirt, oil and surface
saprophytes.
2. Same procedure must be done if the specimen is a NAIL, but it should be CLIP and needs
to be FINELY MINCED before inoculating to media.
3. HAIRcan be obtained by PLUCKING,BRUSHING, or with sticky tape.
4. Normal STERILE must be done if the specimen is a BODY FLUID.

SPECIMEN TRANSPORT AND HANDLING

1. HAIR AND NAIL sent in a DRY envelope, inside a proper container. Other specimens
are usually sentFROZEN or on DRY ICE. Specimen must be inside a packaging with
BIOHAZARD regulations.
2. Any growing CULTURES must be on TUBE media, not in plates. ALUMINUM screw-
capped inner with outer cardboard mailing tube is usually the CONTAINER of fungal
specimen.
3. Inside labeling information must contain: Patient ID, specimen source & suspected
organism
Outside labeling information must state, WARNING: POTENTIAL PATHOGEN.
This labeling format still depends on the protocol of every laboratory.

SPECIMEN PROCESSING

SPECIMEN PROCESSING
Direct exam following KOH
SKIN, NAIL & HAIR

PLEURAL FLUID, Collected fresh due to possible growth of saprophytes

SPUTUM & BRONCHIAL over pathogens such as h. Capsulatum
ASPIRATION

GASTRIC WASHING Same as pleural fluid

GENITO – URINARY First morning specimen

SPECIMENS
 BLOOD/ BONE Inoculated directly to BHI broth and BHI slant
MARROW

WOUND ABSCESS OR Cultured anaerobically, especially if actinomycosis is

DRAINAGE suspected

Centrifuged, examine sediments microscopically then

CSF inoculate

Examine of pus, caseous material or granules; minced

TISSUE SPECIMENS aseptically can use small sterile saline and supernatant
inoculated

LABORATORY DIAGNOSIS OF FUNGI

Fungi can be identified by Directly observing their distinctive MORPHOLOGICAL features on
direct MICROSCOPIC exam of specimen. MedTechs usually look for SPORES, HYPHAE,
MYCELIAL elements, BUDDING yeast and mycotic GRANULES.
1. WET MOUNT PREPARATION
: Good for YEAST examination, loss of FRAGILE structure is minimized
2. 10% KOH mount
: Good for SKIN scrapings, HAIR, nails, SPUTUM, VAGINAL specimens etc. KOH
clears the specimen’s TISSUE cells, mucous without destroying the CELL WALL so fungal
elements can be seen

3. Fungal stains
: DIRECT examination can be aided by the use of STAINS /dyes that can enhance
the VISUALIZATIONS structures
4. Fungal Culture
: Culture is more SENSITIVE than direct EXAM and a portion of the specimen used
in microscopy should be CULTURED.
: Cultures must be held for 21 days at room temperature (25-30 DEGREE CELSIUS).
Yeast grows better at 37 DEGREE CELSIUS and Molds at 30 DEGREE CELSIUS.

FUNGAL STAINS

STAINS USES
LACTOPHENOL COTTON Quick evaluation of fungal structure, stains chitin in
BLUE (LPCB) cell walls of fungi
Periodic acid – Schiff stain Stains polysaccharide in the cell walls of fungi, fungi
stain purple – red with blue nuclei
Gomori Methenamine Silver Outlines fungi in black due to silver precipitating on
stain the fungi cell wall. Internal structure are deep lose to
black; background is light green
GRIDELY STAIN Hyphae and yeast stain dark blue or rose. Tissue
stain deep blue and background is yellow
MAYER MUCICARMINE Stains capsules of cryptosporidium neoformans deep
STAIN rose
FLUORESCENT ANTIBODY Simple, sensitive and specific. Applicable for many
STAIN different fungi
PAPANICOLAU STAIN Good for initial differentiation of dimorphic fungi.
Works well on sputum smears
GRAM STAIN Fungi are gram positive

GIEMSA STAIN Used on blood and bone marrow specimens

INDIA INK Demonstrates capsule of cryptococcus neoformans in

CSF specimens.

FUNGAL CULTURE MEDIA

MEDIA USE
Isolation and preliminary identification of C.
BIRDSEED AGAR neoformans. Appears black colonies

Culture of chlamydoospores – bearing candida

CORNMEAL AGAR albicans

Improves the pigment production of differentiate

CORNMEAL AGAR W/ 1% trichophyton rubrum and trichiphyton mentagrophytes
DEXTROSE
DIFFERENTIAL

Used to isolate Aspergillus spp.

CZAPEK’S AGAR
ENRICHED AND SELECTIVE
MEDIA
Isolate dermotophytes from cutaneous specimens
DERMATOPHYTES TEST
MEDIUM

Selective media used to isolate pathogenic fungi,

MYCOSEL/MYCOBIOTIC dermatophytes and systemic fungi. Contains
AGAR cycloheximide which suppresses growth
SELECTIVE

Contains glucose and modified peptone ph 7,

SABOURAUD DEXTROSE supports growth of fungi and restricts groth of
AGAR (SDA) bacteria
SELECTIVE, G
MISCELLANEOUS FUNGAL TESTS
 1. Germ Tube test

 An important INITIAL STEP in the identification of yeast isolates

 Yeast colonies incubated with SERUM @ 37 deg C

 Confirmatory for the identification of C. albicans

 2. L-DOPA Ferric Citrate Test

 rapid identification of C. neoformans (phenol oxidase)

 + BLACK_
 3. Urease Test

 + result ALKALINIZATION of media indicated by PINK color

 + C. neoformans

 differentiates T. mentagrophytes UREASE POSITIVE from T. rubrum UREASE

NEGATIVE

 4. Hair Baiting Test

 differentiates T.mentagrophytes urease+ from T. rubrum -

 V SHAPED penetration of hair shaft

OTHER IMPORTANT STAINS AND MOUNTS

  1. Temporary Mount

 1.1 10 – 20% KOH

 Dissolves tissue material so fungal elements maybe more visible

 Dissolves KERATIN

 A rapid method of detecting fungal elements

 Gentle HEAT increases rate of CLEARING

 1.2 Lactophenol Cotton Blue (LPCB)

 Preserves and STAINS fungi; excellent MOUNTING media for fungi

 1.3 India ink/Nigrosin

 To demonstrate CAPSULE of C. neoformans

 1.4 Calcofluor White Stain

 Visualization of fungal elements using FLUORESCENCE Microscopy

 Fungal elements will typically appear BLUISH WHITE against a DARK background
2. PERMANENT MOUNTS
2.1 Gram Stain
• Particularly useful for the detection of YEAST
• Most yeast stain partially or completely GRAM POSITIVE & differentiated from
bacteria due to their large size
2.2 Giemsa or Wright’s
• Useful in suspected cases of HISTOPLASMA
• Demonstrates YEAST CELLS within MACROPHAGE
2.3 Periodic Acid Schiff
• Fungal HYPHAE PAS +
2.4 Gomori’s Methenamine Silver
• provides high CONTRAST with MINIMAL background staining
2.5 H& E
• to determine if fungi is hyaline (COLORLESS) or DEMATIACEOUS ( naturally
pigmented)
2.6 Mayer’s Mucicarmine
• demonstrates mucoid capsule of C. neoformans
2.7 Fontana Masson
2.8 Acid Fast / Kinyoun’s
• to differentiate NOCARDIA from Actinomyces, NOCARDIA is slightly AF