MYCV 311 (LABORATORY)
Direct Microscopic Examination
of Fungal Specimen
L. P. CEZAR, RMT
FUNGI
• Fungi are eukaryotic microorganisms and may be yeasts or molds
• Fungi may cause mild infections to life-threatening disease
• Fungal cell wall are made of chitin and they must absorb nutrients from the
environment
• Over 100,000 fungal species are named and there is an estimated 1 to 10 million
undiscovered species
• Only 100 to 150 species are generally recognized as causes of human disease and
approximately 25 species cause the majority of human disease
• Saprobes/Environmental saprobes – fungi that live on nonliving material or dead
organic material; generally nonpathogenic or only causes opportunistic infections to
humans.
YEASTS
YEASTS (Macroscopic Appearance)
• Appearance: colony may appear white to cream
or tan, some may appear pink to salmoncolored.
• Texture: may be mucoid (can flow across the
culture plate), butter-like, velvety or wrinkled.
• Mucoid colonies may be observed in
Cryptococcus spp., trait shared by some
bacterial isolates, such as Klebsiella spp.
Yeasts (Microscopic Appearance)
• Round to oval in shape
• Unlike molds, they do not possess aerial and
vegetative hyphae
• Generally, yeasts reproduce asexually by blastoconidia
formation (budding).
• Budding process involves lysis of the yeast cell wall
so that a blastoconidium (daughter cell) can form.
• Daughter cells exhibit a frail structure known as the
birth scar, while mother cells display a more
persistent bud scar.
Yeast Cells under the Microscope
(Singe and Budding)
Clinical Infections
• thrush
• urinary yeast infection
• meningitis
• pulmonary disease
• septicemia
• moniliasis/candidiasis
• mycotic vulvovaginitis
• endocarditis, etc.
MOLDS
Molds (Macroscopic Appearance)
• have a fuzzy or woolly appearance because of the formation of mycelia
(tube-like structures made up of many hyphae)
• Aerial mycelia – extend above the surface of the colony and gives the fuzzy
appearance ; give rise to fruiting bodies from which asexual spores are
borne.
• Vegetative mycelia – comprise the body of the fungus and extend
downward the medium to absorb nutrients
Molds (Microscopic Appearance)
• Microscopic appearance of molds aids in their identification.
• Hyphae – tubelike projections of fungi. They are usually perforated to allow the
flow of cytoplasm and organelles between cells.
• Antler hyphae - swollen, branching tips that resemble moose antlers
• Racquet hyphae - contain enlarged, club-shaped areas
• Spiral hyphae – tightly coiled
• Rhizoid – root-like structures and might be seen in some Zygomycetes
• Mycelium – root-like structure consisting of intertwined and branching hyphae
Molds (Microscopic Appearance)
• Septate Hyphae - show frequent cross-walls occurring perpendicularly to the outer
walls of the hyphae
• Sparsely septate - have few cross-walls at irregular intervals
• Aseptate – described as absence of septations and has historically been used to
describe the hyphae of the Zygomycetes but they often reveals occasional septations,
therefore more correctly termed as sparsely septate
• Pseudohyphae - not an actual hyphae. It is a chain of yeast cells that remain
attached to one another after budding, resembling hyphae but lacking true septations.
Hyaline and Phaeoid Hyphae
• Hyaline hyphae are nonpigmented or lightly
pigmented.
• Phaeoid (dematiaceous) hyphae are darkly
pigmented due to the presence of melanin in the
cell wall.
• Amount of melanin present will determine the
appearance of the hyphae (pale to dark brown or
almost black)
• Gomori methylene stain - make all fungal
elements appear black.
• Fontana-Masson stain - stains specifically
melanin, causing Phaeoid hyphae to appear
brown, whereas hyaline hyphae stain pink to
red.
Phylum Zygomycota
Phylum Ascomycota
Phylum Basidiomycota
Phylum Deuteromycota
Dimorphic Fungi
• Dimorphism – fungi are able to exist in yeast or mold form, depending on growth conditions
• Yeast phase is seen in vivo or when the organism is grown at 37° C with increased CO2
• Mold phase is seen when the organism is grown at room temperature (22° to 25° C) in
ambient air conditions.
• Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum var. capsulatum,
Paracoccidioides brasiliensis, Sporothrix schenckii, and Penicillium marneffei
Polymorphic Fungi
• Polymorphism – fungi are able to exist in yeast and mold form in the same culture
• This characteristic occurs despite growth conditions
• Exophiala spp. – polymorphic fungi, wherein yeast phase is observed initially, followed by mold
phased as the colony ages.
Direct Microscopic Examination of Fungal Specimen
•KOH (Slide Method)
•KOH (Tube Method)
•KOH with Calcofluor White
•India Ink
•LPCB
•Cellophane Tape Preparation
•Tissue Stains
Direct Microscopic Examination of Fungal Specimen
•Provides rapid result which leads to early initiation of treatment
•Provides a clue to the genus of the organism and gives idea what type of culture
media will be used
•May provide evidence of infection despite negative cultures (patients who are on
antifungal therapy)
•C. albicans - important cause of hospital-acquired infections, especially in an
immunocompromised patient
•Wet preparations – KOH, India ink, and calcofluor white
•Tissue specimen – Histologic Stains
Potassium Hydroxide (KOH Preparation)
•KOH, usually in 10-20% concentration, is useful for
detecting fungal elements embedded within skin, hair, nails,
and tissue
•KOH breaks down the keratin and skin layers to increase
visibility of fungal elements
•Lyses other cells such as epithelial cells, WBCs and RBCs
KOH Preparation (Specimen Collection)
•Skin – clean area with 70% alcohol ; scrape with a sterile blunt scalpel and place
in a petri dish or directly into a microscope slide
•Scalp/Hair – examine the basal portion of the infected hair, collect using a sterile
tweezers or scape the scalp using a blunt scalpel
•Nails – clean area with 70% alcohol and deeply scrape the infected area
KOH Preparation (Transport)
Collect the specimen and properly label with:
•Patient’s name
•Hospital ID number
•Specimen site
•Date and time of collection
•Initials of the person who collected the specimen
KOH Preparation (Reagent)
•10% KOH is commonly used.
•10% glycerol may be used to prevent drying of the KOH solution.
•Modifications of the KOH test - incorporate dimethyl sulfoxide
(DMSO) and a stain into the KOH solution.
•DMSO facilitates more rapid breakdown of cellular debris,
therefore, heat is not required.
•The concentration of the solution should be increased when
testing for thicker specimens.
•Hair (not >5 mm long) and thin skin usually clears within 5 to
15 minutes..
•Skin scales and crusts usually clears within 20 to 30 minutes.
•Nail clippings clears after several hours.
KOH Preparation (Slide Method Procedure)
1. Add 1 drop of KOH solution to the glass slide.
2. Nail scrapings, hair, skin scales, thin slices of tissue, or any specimen for testing is
added to the drop of KOH.
3. Add coverslip.
4. Gently heat the slide for few seconds, and allow to cool for approximately 15
minutes.
5. Examine under the microscope.
KOH Preparation (Tube Method)
1. Specimen is homogenized and placed in a test tube with KOH
Solution
2. Incubate the tube overnight, at 37°C
3. Slide preparation is prepared the next day and observed under
the microscope.
KOH with Calcofluor White
•A drop of calcofluor white (a fluorescent dye)
can be added to the KOH preparation before
adding a cover slip.
•Calcofluor white binds to polysaccharides
present in the chitin of the fungus.
•Fungal elements fluoresce apple green or bluewhite.
•Observed under ultraviolet light in a fluorescent
microscope.
India Ink or Nigrosin
•used to examine CSF for the presence of C.
neoformans
•Centrifuge the specimen and mix the sediment with a
drop of India ink.
•Examined in HPO (x400)
•Budding yeast surrounded by a large clear area against
a black background is presumptive evidence of C.
neoformans.
•Careful examination is necessary since WBCs and other
artifacts may resemble encapsulated organisms.
•Latex agglutination test for cryptococcal antigen in
place of the India ink examination is used nowadays
Lactophenol Cotton Blue (LPCB)
•Used to stain the cell walls of medically important
fungi grown in slide culture
a. Lactic Acid – preserves the morphology of the
fungal structure/elements
b. Phenol – acts as fungicidal that will kill any live
organisms/disinfectant
that
prevents
contamination
c. Cotton Blue – Stains the chitin in the fungal
cell wall
Lactophenol cotton blue–stained slide of macroconidia and
hyphae of the fungal dermatophyte Microsporum gypseum.
Lactophenol Cotton Blue (LPCB) Procedure
1. Sterilize and label the glass slide
2. Place a drop of Lactophenol Cotton Blue Stain in the center of the slide
3. Remove a fragment of the fungus from the fruiting zone using an inoculating or teasing
needle.
4. Place the fragment in the drop of stain and tease gently.
5. Apply a coverslip. Do not push down or tap the cover slip as this may dislodge the conidia
from the conidiophores.
6. Examine the preparation under low and high magnification for the presence of characteristic
mycelia and fruiting structures.
7. Consult appropriate references for diagnostic features of fungi isolated in clinical and non
clinical specimens..
LPCB Cellophane Tape Procedure
1. Place a drop of lactophenol aniline blue stain on a microscope slide.
2. Press the sticky side of unfrosted, clear cellophane tape gently but firmly to the surface
of the colony, picking up a portion of the aerial mycelium.
3. Stick one end of the tape to the surface of the slide adjacent to the drop of stain.
4. Stretch the tape over the stain, gently lowering it so that the mycelium becomes
permeated with stain.
5. Pull the tape taught and stick the opposite end to the glass, avoiding as much as possible
the trapping of air bubbles.
6. Your preparation is now ready for examination.
7. Examine the preparation under low and high magnification for the presence of
characteristic mycelia and fruiting structures.
8. Consult appropriate references for diagnostic features of fungi isolated in clinical and non
clinical specimens..
Tissue Stains
•Tissue stains are used in Histology department for
detection of fungal elements
a. Giemsa Stain - detects H. capsulatum in blood
or bone marrow
b. Periodic acid–Schiff (PAS) - attaches to
polysaccharides in the fungal wall and stains
fungi pink/magenta
c. Fontana-Masson – stains melanin in the cell
wall and identifies the presence of phaeoid
fungi.
Thank You!
REFERENCES:
•
Textbook of Diagnostic Microbiology (5th edition) by Mahon et al
•
Bailey and Scott’s Diagnostic Microbiology (12th edition)