Laboratory Diagnosis of Fungal Infections
maheenmazhar021
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Mar 04, 2025
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About This Presentation
This presentation provides a detailed overview of the laboratory diagnosis of fungal infections, covering specimen collection, direct microscopy, fungal culture techniques, and key identification methods. Designed for medical students, microbiologists, and healthcare professionals, this resource exp...
Slide Content
LABORATORY DIAGNOSIS OF
FUNGUS
By: Dr. Maheen Mazhar Ali
FUNGUS
By: Dr. Maheen Mazhar Ali
LEARNING OBJECTIVES
At the end of this session, students should be able to:
Correlate direct microscopy methods for diagnosing
fungal infections.
Identify fungal infections via culture method.
Differentiate between septate hyphae, nonseptate
hyphae and pseudohyphae.
At the end of this session, students should be able to:
Correlate direct microscopy methods for diagnosing
fungal infections.
Identify fungal infections via culture method.
Differentiate between septate hyphae, nonseptate
hyphae and pseudohyphae.
FUNGI
Molds, yeast, mushrooms, and toadstools are
examples of spore-producing organisms that feed
on organic debris and are known as fungi.
To establish or confirm the diagnosis of a fungal
infection, skin, hair, and nail samples are obtained
for microscopy and culture (mycological
examination).
Molds, yeast, mushrooms, and toadstools are
examples of spore-producing organisms that feed
on organic debris and are known as fungi.
To establish or confirm the diagnosis of a fungal
infection, skin, hair, and nail samples are obtained
for microscopy and culture (mycological
examination).
COLLECTION OF SPECIMEN FOR
FUNGAL TESTING
Fungal microscopy and culture specimens are
transported to the laboratory in a sterile container.
They include:
FUNGAL TESTING
Fungal microscopy and culture specimens are
transported to the laboratory in a sterile container.
They include:
COLLECTION OF SPECIMEN FOR
FUNGAL TESTING
Scrapings of scale, best taken from the leading
edge of the rash after the skin has been cleaned
with alcohol.
Skin stripped off with adhesive tape, which is then
stuck on a glass slide.
FUNGAL TESTING
Scrapings of scale, best taken from the leading
edge of the rash after the skin has been cleaned
with alcohol.
Skin stripped off with adhesive tape, which is then
stuck on a glass slide.
COLLECTION OF SPECIMEN FOR
FUNGAL TESTING
FUNGAL TESTING
COLLECTION OF SPECIMEN FOR
FUNGAL TESTING
FUNGAL TESTING
COLLECTION OF SPECIMEN FOR
FUNGAL TESTING
Hair which has been pulled out from the roots.
Brushings from an area of scaling in the scalp.
Nail clippings or skin scraped from under a nail.
FUNGAL TESTING
Hair which has been pulled out from the roots.
Brushings from an area of scaling in the scalp.
Nail clippings or skin scraped from under a nail.
COLLECTION OF SPECIMEN FOR
FUNGAL TESTING
A skin biopsy.
A moist swab from a mucosal surface (inside the
mouth or vagina) in a special transport medium.
FUNGAL TESTING
A skin biopsy.
A moist swab from a mucosal surface (inside the
mouth or vagina) in a special transport medium.
LABORATORY DIAGNOSIS
METHODS
1. DIRECT MICROSCOPY:
KOH (Potassium Hydroxide) Preparation: Commonly
used to detect fungal elements in skin scrapings,
hair, and nails.
Calcofluor White Stain: Fluorescent dye for fungal
cell walls, used for direct examination.
METHODS
1. DIRECT MICROSCOPY:
KOH (Potassium Hydroxide) Preparation: Commonly
used to detect fungal elements in skin scrapings,
hair, and nails.
Calcofluor White Stain: Fluorescent dye for fungal
cell walls, used for direct examination.
LABORATORY DIAGNOSIS
METHODS
2. CULTURE METHODS:
Sabouraud Dextrose Agar: It is the most commonly
used media in diagnostic mycology.
METHODS
2. CULTURE METHODS:
Sabouraud Dextrose Agar: It is the most commonly
used media in diagnostic mycology.
DIRECT MICROSCOPY OF SKIN
SCRAPINGS AND NAIL CLIPPINGS
The material is examined by microscopy by one or
more of these methods:
Potassium hydroxide (KOH) preparation.
Fluorescent staining.
An unstained wet-mount.
A stained dried smear.
Histopathology of with special stains, such as
periodic acid-Schiff (PAS).
SCRAPINGS AND NAIL CLIPPINGS
The material is examined by microscopy by one or
more of these methods:
Potassium hydroxide (KOH) preparation.
Fluorescent staining.
An unstained wet-mount.
A stained dried smear.
Histopathology of with special stains, such as
periodic acid-Schiff (PAS).
POTASSIUM HYDROXIDE (KOH)
PREPARATION
Skin scrapings and plucked hair samples
(Keratinized tissue specimens).
Treated with 20% KOH which digests the keratin
material release fungal hyphae clearly seen under
the microscope.
Glycerol (10%) is added to prevent drying.
DMSO (dimethyl sulfoxide)- can be added which
helps in tissue digestion.
PREPARATION
Skin scrapings and plucked hair samples
(Keratinized tissue specimens).
Treated with 20% KOH which digests the keratin
material release fungal hyphae clearly seen under
the microscope.
Glycerol (10%) is added to prevent drying.
DMSO (dimethyl sulfoxide)- can be added which
helps in tissue digestion.
POTASSIUM HYDROXIDE (KOH)
PREPARATION
Place the appropriate specimen on a clean glass
microscope slide.
Add one drop of 20% KOH.
Place a cover slip on top of the slide preparation
and press down or tap 5 or 6 times to eliminate air
bubbles. Tilting one end of the slide up, about 10
degrees, while pressing or tapping may assist in
removing bubbles.
PREPARATION
Place the appropriate specimen on a clean glass
microscope slide.
Add one drop of 20% KOH.
Place a cover slip on top of the slide preparation
and press down or tap 5 or 6 times to eliminate air
bubbles. Tilting one end of the slide up, about 10
degrees, while pressing or tapping may assist in
removing bubbles.
POTASSIUM HYDROXIDE (KOH)
PREPARATION
Examine it under a microscope using the low-power
(10x) objective to check for the presence of fungal
elements, such as hyphae or yeast.
If suspect structures are observed, use the high-
power (40x) objective to confirm the presence of
fungi.
The observation of hyphae, budding yeast, or
pseudohyphae constitutes a 'positive' KOH
preparation for fungus.
PREPARATION
Examine it under a microscope using the low-power
(10x) objective to check for the presence of fungal
elements, such as hyphae or yeast.
If suspect structures are observed, use the high-
power (40x) objective to confirm the presence of
fungi.
The observation of hyphae, budding yeast, or
pseudohyphae constitutes a 'positive' KOH
preparation for fungus.
POSITIVE FUNGAL KOH
Dermatophyte: Look for hyaline, septate filaments
with diameters of 5 to 7 µm. This filament is linear
and occasionally branches.
Candida (yeast): Look for round to oval budding
cells as well as "stretched out" budding cells
(pseudohyphae).
Malassezia: Look for short, hyaline, septate hyphae
as well as round, clustered ("spaghetti and
meatballs").
Dermatophyte: Look for hyaline, septate filaments
with diameters of 5 to 7 µm. This filament is linear
and occasionally branches.
Candida (yeast): Look for round to oval budding
cells as well as "stretched out" budding cells
(pseudohyphae).
Malassezia: Look for short, hyaline, septate hyphae
as well as round, clustered ("spaghetti and
meatballs").
POSITIVE FUNGAL KOH
POSITIVE FUNGAL KOH
GRAM'S STAIN OF C. ALBICANS
WET MOUNT OF C. ALBICANS
STAINED DRIED SMEAR
The smears were air-dried, fixed in methanol, and
stained with Giemsa for 20 minutes.
The slide was then rinsed with distilled water, dried,
and observed under direct microscopy to identify
the presence of:
The smears were air-dried, fixed in methanol, and
stained with Giemsa for 20 minutes.
The slide was then rinsed with distilled water, dried,
and observed under direct microscopy to identify
the presence of:
STAINED DRIED SMEAR
Fungal hyphae (branched filaments) making up a
mycelium.
Arthrospores (broken-off spores).
Arthroconidia (specialised external spores).
Spores inside a hair (endothrix) or outside a hair
(ectothrix).
Fungal hyphae (branched filaments) making up a
mycelium.
Arthrospores (broken-off spores).
Arthroconidia (specialised external spores).
Spores inside a hair (endothrix) or outside a hair
(ectothrix).
FUNGAL CULTURE
Identification of Fungi: Culture helps in identifying
fungal species by their colony morphology, color,
and texture.
Culture Duration: Some fungi may take several days
to grow (e.g., dermatophytes) or weeks (e.g.,
dimorphic fungi).
Limitations: Contamination, slow growth, and
inability to identify all species.
Identification of Fungi: Culture helps in identifying
fungal species by their colony morphology, color,
and texture.
Culture Duration: Some fungi may take several days
to grow (e.g., dermatophytes) or weeks (e.g.,
dimorphic fungi).
Limitations: Contamination, slow growth, and
inability to identify all species.
FUNGAL CULTURE
The fungus is incubated at 25–30°C for growth. The
specimen is inoculated into a medium such as
Sabouraud's dextrose agar containing cycloheximide
and chloramphenicol. Cycloheximide is omitted if a
mold requires identification.
The fungus is incubated at 25–30°C for growth. The
specimen is inoculated into a medium such as
Sabouraud's dextrose agar containing cycloheximide
and chloramphenicol. Cycloheximide is omitted if a
mold requires identification.
SABOURAUD DEXTROSE AGAR
Selective medium used primarily for the isolation of
dermatophytes but other fungi and yeasts can also
grow.
Selective medium used primarily for the isolation of
dermatophytes but other fungi and yeasts can also
grow.
SABOURAUD DEXTROSE AGAR
Mycological peptone (enzymatic digestion of
casein)==== Nutritious source of amino acids for
fungal growth.
1.
Dextrose ==== Carbon and energy source.2.
Agar ==== Solidifying agent.3.
Antibiotics (Tetracycline, Chloramphenicol and
Gentamycin) ==== Growth suppression of gram-
positive and gram-negative bacteria.
4.
Mycological peptone (enzymatic digestion of
casein)==== Nutritious source of amino acids for
fungal growth.
1.
Dextrose ==== Carbon and energy source.2.
Agar ==== Solidifying agent.3.
Antibiotics (Tetracycline, Chloramphenicol and
Gentamycin) ==== Growth suppression of gram-
positive and gram-negative bacteria.
4.
GERM TUBE TEST
Also called Reynolds Braude phenomenon.
Used primarily for the presumptive identification of
Candida albicans.
Rapid method of identifying Candida albicans.
It is a specific test for C. albicans but can also be
positive for C. dubliniensis.
Also called Reynolds Braude phenomenon.
Used primarily for the presumptive identification of
Candida albicans.
Rapid method of identifying Candida albicans.
It is a specific test for C. albicans but can also be
positive for C. dubliniensis.
GERM TUBE TEST
Few colonies are mixed with human or sheep serum
and incubated for 2 hours. Wet mount preparation
is examined under microscope.
Germ tubes are formed, described as long tube like
projections extending from the yeast cell. It is
differentiated from pseudohyphae as there is no
constriction at the origin.
Few colonies are mixed with human or sheep serum
and incubated for 2 hours. Wet mount preparation
is examined under microscope.
Germ tubes are formed, described as long tube like
projections extending from the yeast cell. It is
differentiated from pseudohyphae as there is no
constriction at the origin.
GERM TUBE TEST
METHOD:
Make a very light suspension of a yeast in 0.5-1.0 ml
of sterile serum in a test tube.
1.
The optimum inoculum should be 5º 6º cells/mm²2.
Incubate at 35-37 °C for no longer than 3 hours.3.
Place one drop of the yeast-serum mixture on a
slide with cover-slip. Examine microscopically for
germ tube production at 40X.
4.
METHOD:
Make a very light suspension of a yeast in 0.5-1.0 ml
of sterile serum in a test tube.
1.
The optimum inoculum should be 5º 6º cells/mm²2.
Incubate at 35-37 °C for no longer than 3 hours.3.
Place one drop of the yeast-serum mixture on a
slide with cover-slip. Examine microscopically for
germ tube production at 40X.
4.
GERM TUBE TEST
INTERPRETATION:
Positive: Germ tubes are the beginning of true hyphae
and appear as filaments that are not constricted at
their points of origin on the parent cell. E.g., Candida
albicans and Candida dubliensis.
INTERPRETATION:
Positive: Germ tubes are the beginning of true hyphae
and appear as filaments that are not constricted at
their points of origin on the parent cell. E.g., Candida
albicans and Candida dubliensis.
GERM TUBE TEST
GERM TUBE TEST
Negative: If the filaments are constricted and septate
at their points of origin, they are pseudohyphae, not
germ tubes. For example, this applies to all other
Candida species.
Negative: If the filaments are constricted and septate
at their points of origin, they are pseudohyphae, not
germ tubes. For example, this applies to all other
Candida species.
GERM TUBE TEST
HYPHAE
Most multicellular fungal bodies are made up of
filaments called hyphae. Hyphae can form a network
called a mycelium, which is the body of the fungus.
Most multicellular fungal bodies are made up of
filaments called hyphae. Hyphae can form a network
called a mycelium, which is the body of the fungus.
HYPHAE
WHAT IS SEPTATION?
Multicellular fungi form hyphae, which may be septate
or nonseptate. Septate hyphae have distinct cellular
compartments separated by walls called septa. These
septa allow the fungus to keep nuclei contained in
specific regions. Nonseptate hyphae lack septa,
allowing the contents of the hyphae to move freely.
Multicellular fungi form hyphae, which may be septate
or nonseptate. Septate hyphae have distinct cellular
compartments separated by walls called septa. These
septa allow the fungus to keep nuclei contained in
specific regions. Nonseptate hyphae lack septa,
allowing the contents of the hyphae to move freely.
SEPTATE HYPHAE
Hyphae that have walls
(septa) between the cells
are called septate
hyphae.
Hyphae that have walls
(septa) between the cells
are called septate
hyphae.
SEPTATE HYPHAE
NONSEPTATE HYPHAE
Hyphae that lack walls
between the cells are
called nonseptate or
coenocytic hyphae.
Hyphae that lack walls
between the cells are
called nonseptate or
coenocytic hyphae.
PSEUDOHYPHAE
In a yeast cell, the bud remains attached to the mother
cell, elongates, and undergoes repeated budding to
form chains of elongated cells known as
pseudohyphae.
In a yeast cell, the bud remains attached to the mother
cell, elongates, and undergoes repeated budding to
form chains of elongated cells known as
pseudohyphae.
PSEUDOHYPHAE
PSEUDOHYPHAE
PSEUDOHYPHAE
Pseudohyphae = "Stretched-out" yeast cells stuck
together, with constrictions at junctions.
True Hyphae = Continuous, smooth fungal filaments
that form an integrated network.
Pseudohyphae = "Stretched-out" yeast cells stuck
together, with constrictions at junctions.
True Hyphae = Continuous, smooth fungal filaments
that form an integrated network.
THANK YOU
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