This document discusses various methods used to identify fungal infections, including microscopic, cultural, histopathological, and molecular approaches. Microscopic examination methods include wet mount preparations using potassium hydroxide or calcofluor white stain to visualize fungal structures. Histopathological stains like Gomori methenamine-silver and periodic acid-Schiff are used to identify fungi in tissue sections. Culture allows identification based on fungal morphology and biochemical tests. Molecular methods like PCR are also used for diagnosis. Dermatophytes causing ringworm are identified by their microscopic morphology and growth characteristics.

1. FUNGAL IDENTIFICATION
METHODS
Department of Microbiology and Immunology
Presenter: Japhet Peter
Facilitator: Dr .Manyahi
Venue ; Microbiology Teaching Lab.
Date; 30/11/2022

2. Learning outcomes
• Describe fungal identification methods
• List and describe the laboratory approaches
used for diagnosis of fungal infection
• Compare and Contrast the different fungal
identification methods
• Discuss dermatophytes identification
• Describe Fungal Susceptibility Testing

3. Outline
• Introduction
• Fungal Identification Methods
• Summary
• References

4. Introduction
• Identification methods of fungi is the process of
identifying causative agent of infection to human
between none causative agents of infection by using
several approaches; e.g Physical and Laboratory
• Fungi are members of the group of eukaryotic
organisms that includes microorganisms such as
yeasts, molds and mushrooms
• Different types of fungi can cause infections and
different methods can be used to identify the cause of
infection in medical field.

5. Overview of fungi of Medical importance
 Superficial mycoses:
 2 types: surface and cutaneous mycoses
 Skin, hair & nails.
 Deep mycoses:
 2 types: subcutaneous & systemic mycoses
 Range from a symptomatic infection to fatal
disease
 Opportunistic mycoses;-
mostly systemic involvement

6. Fungal Identification
• Identification of fungal infection is based on
morphological and physiological properties of fungi, also
clinical information of the patient
• Laboratory approaches used on diagnosis of fungal
infection includes;
Direct microscopic examination, Histopathological
methods, culture of the organism, serologic tests,
PCR and other molecular methods

7. Fungal Identification…
• The proper diagnosis of fungi depends on proper
specimen collection by sterile technique including;
– Decontamination of skin surface with 70% ethanol
– Surface scraping to remove skin scales
– Treatment of specimen with 10% potassium hydroxide
to destroy tissue elements

8. Fungal Identification…
• Specimens are supposed to be delivered at clinical
laboratory/histological lab, in a clearly labeled, sterile
containers appropriate to the type of material being
investigated
• The selection of appropriate collection devices &
transport containers, labeling of the specimen &
complete requisition forms are important
considerations in ensuring the correct diagnosis of
fungal infections

9. Fungal specimens for identification
• Specimens include;
 Sputum-for aspergillosis, histoplasmosis,
blastomycosis
 Biopsy material-for Histoplasmosis
 Skin scrapings-Hair or nail clippings,
 blood-for systemic infections
 Vaginal swab-for candidiasis
 Body fluids eg CSF for Cryptococcosis
exudates from granulous or ulcerative
lesions

10. Fungal Identification Methods
1. Macroscopic examination eg Wood’s lamp test
2. Microscopic examination
a) Wet preparation (Mount)-KOH,India ink
b) Staining smears (Gram stain,giemsa stain)
c) Histopathological methods (H&E,PAS)
3. Culture methods
4. Molecular methods
5. Biochemical methods
6. Immunological methods

11. Microscopy
• It is Rapid method and very crucial in the diagnosis
of fungal infections, (superficial and subcutaneous)
• Detects; asexual spores, hyphae, or yeasts using
light microscope.
Examples:- capsule of Cryptococcus neoformans,
Candida albicans etc

12. Microscopy
1.Wet mount preparations - Potassium hydroxide (KOH)
• Slide Method procedure;
– Specimens are placed on a slide containing a drop of
10–20% potassium hydroxide.
– Cover with cover slip, examine immediately after 20
minutes using light- microscope by using 10x
objective.

13. Microscopy
• Tube Method
– Mainly prepared for biopsy specimens, which take
longer time for dissolution
– The homogenized biopsy tissue is dissolved in
10% KOH and examined after keeping for
overnight incubated at 370C.

14. Microscopy
Interpretation of the results;-
• From skin or nails specimens ; branching hyphae or
chains of arthroconidia (arthrospores) are seen easily
• From hairs specimen; most microsporum species form
dense sheaths of spores around the hair (ectothrix)
Trichophyton tonsurans and Tricholosporum violaceum
producing arthroconidia inside the hair shaft (endothrix)

15. KOH-wet mount
Spores outside a hair (ectothrix) Spores inside a hair (endothrix

16. Microscopy
2. Calcofluor white stain;
This is nonspecific fungal cell wall fluorescent dye stain
which is used for identification of fungi in tissue
specimens under fluorescent microscope. Fungal elements
in exudates and small skin scales appears;
Fluorescent bright green to blue and white appearance.
See procedure at next slide

17. Calcofluor white stain procedure
1.Put the sample onto a clean glass slide
2.Add 1 drop of calcofluor white stain
3.Place a coverslip over the sample
4.Allow to stand for 1 minute
5.Examine the slide under uv light at 355nm
wavelength.
Interpretation of results;
Fungal or parasitic organisms appear-
fluorescent bright light green to blue, other--

18. Microscopy

19. Microscopy-----
3. Lactophenol cotton blue stain; is used to
determine the morphology of the conidiogenous
cells and the conidia that identify filamentous
fungus
Procedure;
1. Plae 2-3 drops of Lactophenol Cotton blue stain on
the slide.
2. Add fungal culture to the drop of the stain
3. Carefully tease fungal culture using forcep as to
prepare a thin preparation

20. 4 . Place a coverslip on the preparation
5. Allow to stand for 5 minutes
6.Observe microscopically using 100x objective
Interpretation of results;
-Fungal structures such as hyphae and
spores are stained blue in color.

21. Microscopy…
Aspergillus fumigatus
Mould

22. Indian ink for C.neoformans
wet mount
. preparation of cerebrospinal fluid (CSF),
highlights Cryptococcus neoformans
capsule (method-insensitive 50% )

23. Microscopy
5. Gram Stain; detection of
yeasts such as Candida or
Cryptococcus,
Fungi such as Aspergillum,
Intercellular yeast forms such
as Histoplasma capsulatum in
Wright stain for;
peripheral blood smear, bone
marrow or tissue
• Yeast cells

24. Histopathological methods
• Examination of tissue sections in diagnosis of
subcutaneous and deep fungal infections
1. Hematoxylin and Eosin (H&E) stain; routine
histological stains but many fungi stain poorly
–Asteroid body consists of a central basophilic
yeast cell surrounded by radiating extensions of
eosinophilic material (depositions of antigen-
antibody complexes and complement)

25. H&E stain
Endospores and sporangia
of Rhinosporidium seeberi
Blastomyces dermatitidis

26. Histopathological…
2. Gomori methenamine-silver stain
(GMS); fungi stained dark gray to
black
3. Mayer’s mucicarmine stain;
specifically used to show capsular
material of Cryptococcus, endospores
and sporangia of Rhinosporidium
seeberi
GMS-Stain
Cryptococcus neoformans: Pleomorphic
yeast-like cells and formation of narrow-
based buds are typical. The encapsulated
strains have capsular material detected with
mucin stain.

27. Histopathological…
4. Gridley fungus stain; fungi stained purplish
rose with a yellow background
5. Calcofluor white stain; used on tissue
sections, fungi fluorescent blue white
appearance on a dark background
6. Periodic acid-Schiff (PAS); fungi stained hot
pink to red based on the presence of chitin and
polysaccharide in fungi cell wall

28. Histopathological…
–Detect non-pigmented branching, septate
hyphae is typical of Aspergillus infection
Aspergillus -skin biopsy

29. Histopathological…
7. Direct immunoflourescence
–Immunoperoxidase and immunofluorescent
staining reagents, detects monoclonal and
polyclonal for some fungi
–Immunochemical staining, facilitate the
identification of atypical fungal elements and
assist the diagnosis of mixed infections

30. Histopathological…
8. In situ hybridization
–DNA probes to the nucleic acid of the fungal
agent, directly on the slide (in situ
hybridization) or in a test tube

31. Culture Method
• Specimens; cultured on inhibitory mold agar e.g.,
Sabouraud's dextrose medium (SDA), enriched
media with antibiotics (chloramphenicol ) to
inhibit bacterial growth, and enriched media with
both antibiotics and cycloheximide (which
inhibits many saprophytic fungi). Incubated at
25–35 °C

32. Culture…
• Selective media;-
– Corn meal agar (CMA) suitable for
sporulation, chlamydospore formation by
C.albicans.
– Bird seed agar – cryptococcus, forms brown
colonies
– Brain Heart Infusion (BHI) agar – dimorphic
& other fastidious fungi
• The appearance of the mycelium and the nature
of the asexual spores are frequently sufficient to
identify the organism

33. Culture…
– Identification of yeast cultures
• Morphologic characteristics (presence of capsule,
formation of germ tubes in serum, and morphology
on cornmeal agar)
• Biochemical tests (urease, nitrate reduction, and
carbohydrate assimilations and fermentations)
• Also can be accomplished with DNA probes, which
are available for Cryptococcus

34. Culture…
–Identification of filamentous fungal cultures
• Immunologic criteria; uses an
immunologic method called exoantigen
testing, in which antigens extracted from the
culture to be identified are immuno diffused
against known antisera
-Blood culture should be performed in all cases
of suspected deep fungal infection

35. Fungi on culture plates
• ,
Penicillium
Aspergillus
versicolor

36. Dermatophytes Identification
–Species identified based:
• Colonial morphology (growth rate, surface
texture, and any pigmentation)
• Microscopic morphology (macroconidia,
microconidia)
• In some cases, nutritional requirements
Note:dermatophyte- pathogenic fungus that grows on skin, mucous membranes,
hair, nails, feathers, and other body surfaces, causing ringworm and related disease

37. Dermatophytes Identification…
• Microscopic morphological characteristic (microconidia
and/or macroconidia)
– Specimens inoculated onto inhibitory mold agar or
Sabouraud's agar slants containing cycloheximide
and chloramphenicol to suppress mold and bacterial
growth, incubated for 1–3 weeks at room
temperature

38. Dermatophytes Identification…
Three genera are recognized:
• Epidermophyton: Smooth thin-walled Macroconidia
only present, no microconidia, colonies are green-
brown to khaki colour
• Microsporum: Macroconidia with rough walls
present, microconidia may also be present
• Trichophyton: Microconidia present, smooth-walled
macroconidia may or may not be present

40. Dermatophytes Identification…
• Trichophyton species; Infected hair, skin, or nails,
develop cylindric, smooth-walled macroconidia
• T mentagrophytes- cottony to granular colonies ,
grape-like clusters of spherical microconidia on
terminal branches. Coiled or spiral hyphae are
commonly found in primary isolates

41. Dermatophytes Identification…
• T rubrum-white colonies , cottony surface and a deep
red, no diffusible pigment when viewed from the
reverse side of the colony. The microconidia are small
and piriform (pear-shaped)
• T tonsurans- flat, powdery to velvety colony on the
obverse surface that becomes reddish-brown on
reverse; the microconidia are mostly elongated

42. Dermatophytes Identification…
• Epidermophyton floccosum; The only pathogen in this
genus, infects the skin and nails.
- Produces macroconidia, which are smooth-walled,
clavate, two- to four-celled, and formed in groups of
two or three
-The colonies are usually flat, smooth with a tan to olive-
green tinge

43. Dermatophytes Identification…
• In addition to gross and microscopic morphology, a
few nutritional or other tests, such as growth at 37
°C or a test for in vitro hair perforation, are useful in
differentiating certain species.

44. Dermatophytes Identification…
• Microsporum species; Infect hair and/ or skin,
produce distinctive multicellular macroconidia with
echinulated(small spines) walls.
• M canis -forms white cottony surface colonies
and a deep yellow color on reverse; the thick-
walled, 8- to 15-celled macro conidia frequently
have curved or hooked tips

45. Dermatophytes Identification…
• M gypseum- produces a tan, powdery colony and
abundant thin-walled, four- to six-celled macroconidia

46. FUNGAL SUSCEPTIBILITY
TESTING
• Broth micro dilution method is used for
susceptibility testing
• Antifungal resistance is a particular problem
with Candida infections.
• About 7% of all Candida blood stream isolates
tested at CDC were resistant to fluconazole

47. Biochemical Tests
a. Urease test
–Urease positive e.g.
Cryptococcus neoformans
b. Nitrite reduction
–Fungi that reduce nitrate e.g. Aspergillus spp
c. Carbohydrate fermentation
–C. albicans ferment glucose, galactose and
maltose

48. Molecular Methods
• Direct detection of nucleic acid amplification
– DNA probes used to identify fungi colonies
growing in culture at a much earlier stage , for
detection of Coccidioides, Histoplasma,
Blastomyces, and Cryptococcus
– Other methods includes, the use of
conventional PCR formats, multiplex PCR
(amplify more than one target sequence-
multiple sets primers), pan-fungal PCR, real-
time PCR methods.

49. Molecular Methods…
–These techniques permit quantification of the
amounts of fungal nucleic acid that are present
in clinical samples
• Strain typing

50. Immunological Methods
• Tests for the presence of antibodies
(histoplasmosis, coccidioidomycosis) and
antigens (cryptococcosis, aspergillus, candidiasis,
histoplasmosis) in the patient's serum or spinal
fluid for diagnosing systemic mycoses
• Detection of IgM indicates recent exposure
whereas IgG indicate exposure some time ago

51. Immunological…
• Complement fixation test, used in suspected
cases of coccidioidomycosis, histoplasmosis,
and blastomycosis
• Latex agglutination test, detected the presence
of the polysaccharide capsular antigens of C.
neoformans in the spinal fluid (cryptococcal
meningitis)

52. Summary
Fungus Microscopic
Morphologic
Features in
Clinical
Specimens
Morphologic Characteristic features in Culture Tests for
Identification
Macroscopic Microscopic
Candida Oval budding
yeasts 2-6 µm in
diameter ( hyphae
& pseudohyphae
may present)
Variable; colonies
usual pasty, white to
tan & opaque (may
have smooth or
wrinkle morphology)
Clusters of blastoconidia,
pseudohyphae and/or
terminal chlamydospores in
some spp
Germ tube,
PNA-FISH
Cryptococuss
neoformans
Spherical budding
yeast of variable
size 2-15µm.
Capsule may
present, no
hyphae or
phseudohypae
Colonies are shiny,
mucoid, dome shaped
and cream to tan in
color
Budding spherical cells of
varying size, capsule
present, no pseudohyphae
Urease (+),
phenoloxidase
(+), EIA test for
polyscharide
Aspergillus Septate,
dichromously
branched hyphae
3-6
Varies of species A.
fumigates; blue-green
to gray A. niger; black
Varies of species
Conidiosphores with
enlarged visceral covered
flask-shaped metulae or
philalides. Hyphae are
hyaline and septate
Base on
microscopic and
colonial
morphology
Gene sequence

53. Summary…
Fungus Microscopic
Morphologic
Features in Clinical
Specimens
Morphologic Characteristic features in Culture Tests for
Identification
Macroscopic Microscopic
Histoplasma
capsulatum
Small 2-4 µm
budding yeast within
macrophages
Colonies are slow
growing and white or
buff-brown in
color(25 o
C)
Yeast phase colonies
(37 o
C) are smooth,
white and pasty
Thin septate hyphae that
produce tuberculte
macroconidia and smooth-
walled microcornidia (25
o
C).Small, oval, budding
yeast produced at 37 o
C
Demonstration of
temperature-
regulated
dimorphism by
conversion from
mold to yeast phase
at 37o
C, acid probe
test allow
identification without
phase conversion
Mucomycetes Broad thin-walled,
pauciseptate hypae, 6-
25 µm with
nonparallel sides and
random branches.
Hyphae stain poorly
with GMS stain and
ofen stain well with
H&E stain
Colonies rapidly
growing, wooly and
gray-brown to gray-
black in color
Broad. Ribbon-like hyphae
with rare septa.
Sporangium or sporangial
produced from
sporangiophore. Rhizoids
present in some species
Identification based
on microscopic and
colonial macroscopic
morphology. Gene
sequencing
Dematiaceous
molds
Pigmented ( brown,
tan or black) hypae,
2-6µm wide, May be
branched or un-
branched
Collonies usually
rapidly growing,
woody and gray,
olive, black or brown
in color
Varies depend on genus
and species. Hyphae are
pigmented. Conidia may be
single or in chains, smooth
or rough and dematiaceous
Identification based
on microscopic and
colonial macroscopic
morphology. Gene
sequencing

54. References
• Johnson, Arthur G.; Ziegler, Richard J.; Lukasewycz,
Omelan A.; Hawley, Louise B. in BRS Microbiology and
Immunology, 4th Edition
• Murray Rosential Pfaller in Medical Microbiology,
seventh edition
• http://www.mycology.adelaide.edu.au/virtual/2005/ID2-
Nov05.html
• https://bmcmicrobiol.biomedcentral.com/articles/10.1186/
1471-2180-8-135
• https://www.cdc.gov/fungal/antifungal-resistance.html