Laboratory diagnosis of mycology microscopy, staining techniques, culture media, serology

Laboratory Diagnosis of Mycology:
Microscopy, Staining Techniques,
Culture Media, Serology
UNDER GARADUATE STUDENT’S LECTUER ON
BY
GUNJAL PN
ASSIST. PROF.
DEPT OF MICROBIOLOGY
DVVPF’S MEDICAL COLLEGE & HOSPITAL
AHMENDAGAR
6/16/2021 Dept of Microbiology 1

Competency
• Following are the competencies for this theory class :
• MI 1.1 –The different methods used in detection of fungal
infections. (K/KH/Y) (Lecture, small group discussion)
(Written/Viva)
• MI1.2 – Perform and identify the different causative agents of
infectious diseases by Staining methods for fungal diagnosis.
(GM/ZN/KOH/LPCB/Special stains) – (S/P/Y ) (DOAP)
(Written/Viva /Skill assessment)

• MI 8.9 – Discuss appropriate methods of collection of samples
for lab diagnosis of fungal infections –(K/KH/Y) (Lecture/ small
group discussion) (Written/ Viva voce).
• MI 8.10 – Demonstrate appropriate method of collection of
specimen for lab diagnosis – (S/SH/Y) (DOAP) (Skill
assessment)
• Domain - K- Knowledge / S- Skill.
• Level of competency – K- Knows / KH-Knows How/ S- Shows/ SH- Shows
How/ P- Perform independently.
• Core – Y – Yes (Necessary to complete requirement of subject) / N- Non-
core.

Learning Objectives
At the end of the session, the students will be able to understand:
• Site and type of specimens to be collected for laboratory diagnosis of fungal
infections.
•Microscopic examination for laboratory diagnosis of fungal infections.
•Staining techniques used for laboratory diagnosis of fungal infections.
•Culture Media used for cultivation and identification of fungi in laboratory.
•Serological Methods used in routine laboratory diagnosis of fungal infections .

General Mycology
• Medical Mycology – Branch of Medical Microbiology
deals with study of fungi responsible to cause human
infections and important medically.
• “Fungus” – Word derived from Greek word “Mykes”
meaning mushroom (edible fungus).

Fungi differ from Bacteria
• Fungi are eukaryotic and possess all eukaryotic organelles
such as mitochondria.
• Cell wall is rigid – chitin and Beta-glucans- (is the site of
action for most of antifungal drugs) and other
polysaccharides but not peptidoglycan.
• Thus fungi are resistant to antibiotics as penicillin.
• Cell membrane – Ergosterol- site of action for antifungal drug
like amphotericin B and azole group.
• May be unicellular or multicellular.
• Lack chlorophyll and divide by asexual and/or sexual methods
by producing spores.

Classification of fungi
• MORPHOLOGICAL CLASSIFICATION -
1. Yeast:
• Round to oval cells that reproduce by asexual process of budding in which
cell develops a protuberance which elongates and finally separates from
parent cell.
• Yeast colonies resemble bacterial colonies in appearance and in
consistency.
- Cryptococcus neoformans (pathogenic)
- Saccharomyces cerevisiae (non-pathogenic).
2. Yeast-like:
- Yeast like fungi grow partly as yeast and partly as elongated cells
resembling hyphae . Later form forming pseudohyphae (e.g. Candida).
- Differentiated from true hyphae as they have constrictions at septa.

Morphological Classification -
3. Molds –
• The basic morphological element of filamentous fungi are long
branching filaments of hyphae, which intervene to produce
mass of filaments or mycelium.
• Colonies are strongly adherent to the medium and unlike most
bacterial colonies cannot be emulsified in water.
• The surface of these colonies may be velvety, powdery, or may
show a cottony Arial mycelium.
• Reproduce by forming different type of spores. E.g. –
Dermatophytes, Aspergillus, Penicillium, Mucor, Rhizopus.

• Produce two type of mycelium:
• A. Vegetative Mycelium – Hyphae that
penetrate the supporting medium and absorb
the nutrients.
• B. Aerial Mycelium – Hyphae projects above
the surface of medium and bears the
reporductive structure called Conidia.

4. Dimorphic fungi: exist as molds in the environment at
ambient temperature (25°C) and as yeasts in human
tissues at body temperature (37°C).
 Histoplasma capsulatum
 Blastomyces dermatitidis
 Coccidioides immitis
 Paracoccidioides brasiliensis
 Penicillium marneffei
 Sporothrix schenckii.

Morphological forms of fungi

Taxonomical Classification
• Based on the production of sexual spores
1. Phylum Zygomycota: sexual spores – Zygospores, and
possess aseptate hyphae, e.g. Rhizopus and Mucor.
2. Phylum Ascomycota: Sexual spores - Ascospores and possess
septate hyphae, e.g. Aspergillus.
3. Phylum Basidiomycota: Sexual spores - Basidiospore e.g.
Cryptococcus.
4. Phylum deuteromycota (Fungi imperfecti): Majority of these
are medically important but sexual state is either absent or
unidentified yet. Hence grouped as Fungi Imperfecti. For e.g.
Candida.

Laboratory Diagnosis of Fungal
Disease
• To confirm clinical suspicion to establish fungal
cause of disease.
• To help in -
– Choosing a therapeutic agent
– Monitoring the course of disease
– Confirming mycological cure

Laboratory Diagnosis of Fungal
Disease
• The laboratory diagnosis of fungal diseases
comprises of following points :
• Specimen Collection.
• Microscopy.
• Culture Media.
• Serological / Immunological Methods of
identification.
• Molecular Methods of Identification.
• Other Methods of Identification.

Specimen Collection
• Sites & Types of Specimens:
• Specimen collection depends on the
corresponding disease.
• Very important to proceed for a final
diagnosis.

• Clean the part with 70% alcohol
• Collect the material in a sterile paper or a
sterile petridish to -
– Allow drying of the specimen
– Reduce bacterial contamination
– Maintain viability
(a) Superficial Mycosis

• Dermatophytic lesion – spreads outward in a
concentric fashion with healing in the center – scrape
outwards from the edge of the lesion with a scalpel
blade or use Cellophane tape
• Scalp lesion – scraping with a blunt scalpel, including
hair stubs, scales & contents of plugged follicles.

• Scalp lesion – Wood’s lamp examination of
infected hair – fluorescence- ring worm infection.
Hairbrush sampling technique – esp. for culture.
• Onychomycosis – Fungal infection of nail.
• Stop antifungals one week prior to collection.
• Sample collected from base of the nail as fungus
in distal end is non-viable, include full thickness of
nail.
• Mucosal infections – Mucosal scrapings are
preferred over swabs.

(b) Subcutaneous Mycosis
• Scrapings or crusts from the superficial parts of
lesions.
• Collect carefully usually contaminants are more in
these area.
• Pus aspirates & Biopsy are valuable.
• Biopsy should be avoided in sporotrichosis as it
leads to spread of infection and hinder healing.

(c) Systemic Mycosis
• Pus
• Biopsy
• Feces
• Urine
• Sputum
• CSF
• Blood
• Scrapings or swabs
from the edge of
lesions.

• Proper collection of specimen and in adequate
quantity.
• Early transport to the lab to avoid overgrowth of
contaminant.
• Respiratory specimens
– Sputum – early morning sample, after mouth wash, flakes
to be used for culturing.
– Bronchoscopy – if non productive cough. BAL can be
collected.
– Bronchial brushings or lung biopsy – to rule out invasion
or colonisation.
Collection & Transport of specimen

• Blood
– In biphasic Brain Heart Infusion agar.
– Inoculated in 2 bottles – for dimorphic fungi.
– Subculture is done after 2 days and 7 days.
• Cerebrospinal fluid
– Should be immediately processed else stored at RT or at
30°C in an incubator.
– Centrifuge & use sediment for culture.

• Skin, Hair & Nail
– Taken for Dermatophytic infections
– Hair – plucked with forceps.
• Tissue, BM & Body fluids
– Tissues – grind or mince before culturing.
– Body fluids – centrifuge & use sediment for culture.
• Urine – centrifuge & use sediment for culture.

• Stool – Not suitable. Intestinal biopsy better.
• Eye – In Keratomycosis, scrapings from base
and margins of ulcer are taken using Kimura’s
spatula.
• Aspirates can be taken from hypopyon or
endophthalmitis.

• Direct examination
• Fungal culture
• Serological tests
• Molecular methods
• Other methods
Laboratory Diagnosis

• MICROSCOPY –
• WET MOUNTS
• Potassium Hydroxide (KOH) preparation:
• Keratinized tissue specimens such as skin scrappings and
plucked hair samples are treated with 10% KOH.
• KOH digests keratin – fungal hyphae seen clearly under
microscope.
• Heat the slide gently over the flame and leave it aside for
5-10mins before observing under microscope.
Direct Examination

KOH - Aspergillus
• 10% KOH is usual conc. Used.
• 20-40% KOH used for
specimens such as nails- takes
longer time to dissolve.
• Glycerol (10%) can be added
to prevent drying.
• Dimethyl sulfoxide (DMSO)
used to help in digesting
tissues.
• Interpretation – caution while
reporting hyphae – can be
confused with cotton fiber,
hairs present in specimen.

• Gram stain – fungi are
Gram +ve.
• Useful in detecting –
Candida and Cryptococcus
• – Gram +ve yeast cells.
• India Ink and Nigrosin –
Used as Negative stain to
demonstrate capsule o
Cryptococcus
neoformans.
Direct Examination

• Calcofluor White Stain :
• A fluorescent stain – excellent
morphology of pathogenic fungi seen.
• More sensitive than other stains; binds to
cellulose and chitin of fungal cell wall
present in high conc. and fluoresce under
UV light.
• If supplemented with KOH, useful for
corneal scrapings which has scanty fungal
elements.
Direct Examination

• Histopathology – Useful in demonstration of fungal
elements such as - yeast cells, hyphae,
pseudohyphae, arthrospores, chlamydospores, and
spherules.
• In biopsy tissues.
• Useful in detecting invasive fungal infections.
– Routine stain – Hematoxylin & Eosin (HE) - very useful to
visualize the host's response.
1. Superficial infection – Acute, subacute or chronic
dermatitis with folliculitis.
2. Subcutaneous & systemic infections – Granulomatous
reaction with fibrosis or pyogenic inflammation.
Direct Examination

• HISTOPATHOLOGICAL STAINS –
• PERIODIC ACID SHIFF (PAS) STAIN
• Recommended stain for detecting
fungi.
• PAS positive fungi appear
magenta/deep pink – nuclei – blue.
• GOMORI METHENAMINE SILVER
(GMS) – used as an alternative to PAS
stain for detecting fungi.
• It stains both live and dead fungi.
• As PAS can stain only live fungi.
• GMS stains the polysaccharide
component of the cell wall.
• Fungi appears – Black
• Background tissue – Pale green color.
Direct Examination
Budding Yeast, Pseudohyphae,
Candida spp.
Pulmonary Histoplamosis
mimicking lung carcinoma

• MUCICARMINE STAIN –
• Used for staining the carminophilic cell
wall of Cryptococcus and
Rhinosporidium.
• MASSON FONTAN STAIN -
• It is used for pigmented fungi.
• FLUORESCENT – ANTIBODY STAINING
–
• To detect fungal Antigen in specimens
such as pus, blood, CSF, tissue
sections.
• Advantage – Can detect even when
few organisms are present.
Direct Examination

• LACTOPHENOL COTTON BLUE (LPCB)–
• It is used to study microscopic
appearance of the fungal isolates
grown in culture.
• It contains :
• Phenol acts as disinfectant.
• Lactic acid preserves the morphology
of fungi.
• Glycerol prevents drying.
• Cotton Blue stains the fungal elements
blue.
Direct Examination

Culture Media
• Fungal Culture is frequently performed for isolation and correct
identification of the fungi.
• CULTURE MEDIA –
• SABOURADU’S DEXTROSE AGAR (SDA) –
• Most commonly used medium in diagnostic mycology.
• Contains – Peptone - 1%.
• Dextrose – 4%.
• pH – 5.6.
• Antibiotics (gentamicin, chloramphenicol) and cycloheximide
• This may not support some pathogenic fungi as
• Cycloheximide is not used when Cryptococcus, Aspergillus or
Penicillium is suspected

• CORN MEAL AGAR AND RICE STARCH AGAR –
• They are nutritionally deficient media used for stimulation of
chlamydospores production.
• BRAIN HEART INFUSION AGAR (BHI) AND BLOOD AGAR –
• Enriched medias – used for cultivation of fastidious fungi like –
Cryptococcus & Histoplasma.
• NIGER SEED AGAR & BIRD SEED AGAR –
• Used as selective media for Cryptococcus forms brown color
colonies.
• CHROM AGAR CANDIDA MEDIUM –
• Used as selective as well as differential medium for speciation of
Candida.
Culture Media

Corn Meal Agar
Bird Seed Agar

CHROM Agar
C.tropicalis
C.krusei
C.albicans

• Specimens should be cultured
on agar slants:
– Safe
– Require less space
– More resistant to drying during
prolonged incubation
– Blood cultures should be
inoculated in to biphasic blood
culture bottles
Fungal Culture

• Temperature requirement
– Majority of fungi – 37°C
– Superficial mycosis – 30°C
– Dimorphic fungi – 25°C & 37°C
• Incubation time
– At least 4 weeks
– Usually positive cultures are obtained in 7-10
days
– Candida & Aspergillus - 24 to 72 hrs
Fungal Culture

Interpretation of Fungal Culture
• Isolation of an established pathogen like Histoplasma capsulatum or
Cryptococcus neoformans – Evidence of infection.
• Isolation of commensal or opportunistic fungi like Candida or
Aspergillus -
• Consider following points:
• Isolation of same strain in all culture tubes.
• Repeated isolation of same strain in multiple specimens.
• Isolation of same strain from different sites.
• Immune status of patient.
• Serological evidence – Ag or Ab test confirms presence of specific Ag or
Ab.

Culture identification
• The correct identification of the fungus is based on the macroscopic appearance
of the colonies grown on culture media and microscopic appearance (LPCB
mount).
• MACROSCOPIC APPEARANCE OF COLONIES –
• RATE OF GROWTH – Rapid growth (<5 days): Seen in Saprophytes, Yeasts, and
agents of opportunistic myocses.
• PIGMENTATION –
• Seen on reverse side of the culture media slant.
• TEXTURE –
• Refers to if allowed to touch how colony will feel. For e.g. Waxy/leathery, velvety,
yeast-like, cottony, or granular/powdery.
• COLONY SURFACE –
• Rugose (radial grooves), folded, verrucous or cribriform (Brain like).

• MICROSCOPIC APPEARANCE OF FUNGI –
• SLIDE CULTURE –
• Tedious procedure, but gives the most accurate in situ microscopic
appearance of the colony.
• A sterile slide is placed on a bent glass rod in a sterile petri dish.
• Two square agar blocks measuring around 1 cm2 (Smaller than
coverslip) are placed on slide.
• Bit of fungal colony is inoculated onto the margins (at the centre) of
the agar block.
• Then the coverslip is placed on the agar block and the petri dish is
incubated at 250C.
• After sufficient growth occurs, LPCB mounts are made both from
the coverslip and below the slide.
Culture identification

Colony Morphology
Colony morphology – colour, texture,
pigment
LPCB

• CELLOPHANE TAPE MOUNT –
• The impression are taken by placing the cellophane tape on
the colonies present on the surface of SDA plate.
• LPCB mount is made from cellophane tape.
• This is easy to perform than slide culture technique.
• BIOCHEMICALS –
• Ability to assimilate carbon & nitrogen, sugar fermentation.
• Urease test can be done for the fungi that produces enzyme
Urease – for e.g. Cryptococcus neoformans.
Colony Morphology

• These tests are available to detect the antibody or antigen from
serum and other body fluids.
• ANTIBODY DETECTION –
• Done using ELISA, immunodiffusion test, agglutination test, and
complement fixation test (CFT).
• ANTIGEN DETECTION –
• Various fungal antigens can be detected in clinical specimens such
as blood, CSF, urine, etc.
• Cryptococcal capsular antigen –
• From CSF by latex agglutination test.
• Detection of Aspergillus specific Galactomannan Antigen in
patient’s sera or urine (by ELISA).
Serology & Immunology

• β-d-Glucan Assay –
• Detecting β-d-Glucan antigen in blood by ELISA.
• It is marker of invasive fungal infections, raised in all invasive
fungal infections.
• Except for zygomycosis, blastomycosis and cryptococcosis.
• IMMUNOHISTOCHEMISTRY –
• It refers to detecting antigens (e.g. proteins) on the cells of a tissue
section by using fluorescent tagged antibodies that bind specifically
to the antigens.
• It is useful in deep mycoses.
Serology & Immunology

Automations
• Automated identification systems such as MALDI-TOF
and VITEK are revolutionary in accurate identification
of yeasts and to some extent molds.

Molecular Methods
• Polymerase Chain Reaction and its modifications
such as
• Multiplex,
• Nested
• most advanced Real Time PCR and
• DNA Sequencing
• These methods have been developed for accurate
identification of fungi from culture as well as from
the specimens.

Other Methods of identification
• For Candida –
• Germ tube test – A screening test used to
differentiate Candida albicans from other yeasts and
yeasts like fungi.
• Suspected isolate of Candida spp. is incubated in
human or sheep serum at 370C for 3 hrs.
• If suspected species is Candida albicans the Germ
tube test is positive – they form Germ tube a
filamentous outgrowth appears from yeast cells.

Other Methods of identification
• For Dermatophytes –
• HAIR PERFORATION TEST
• REQUIREMENT –
• Autoclaved blonde pre-pubital hair cut into
short pieces (1cm)
• Sterile distilled water 5 ml in a suitable vial.

• PROCEDURE -
• Place hair in water in vial.
• Inoculate with small fragments of the
test fungus.
• Incubate at room temperature.
• Individual hairs are removed at intervals
up to 4 weeks and examined
microscopically in lactophenol cotton
blue.
• Isolates of T. mentagrophytes produce
marked localised areas of pitting and
marked erosion whereas those of T.
rubrum do not.

Expected Questions
• Long Answer Questions
• Describe and discuss the methods for laboratory
diagnosis of fungal infections.
• Describe in details the culture media used for
fungal diagnosis.
• Describe the Immunological methods used for
the fungal diagnosis.

Expected Questions
• Short Answer Questions
• Discuss the serological diagnostic methods used in fungal
laboratory diagnosis.
• Describe the culture media used for diagnosis of fungal
infections.
• Describe the Molecular methods used for fungal diagnosis.
• Describe the stains and staining methods used in fungal
diagnosis.

MCQ
• 1. In the LPCB stain Phenol act as ………
• A. Mordent
• B. Stain
• C. Disinfectant
• D. Decolouriser
• Ans: C
• 2. Corn Meal Agar is ……………….. Medium.
• A. Nutritionally deficient
• B. Nutritionally healthy
• C. Basal
• D. Selective
• Ans : A

Thank you all !

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