MYCOLOGY

SPECIMEN COLLECTION
& HANDLING
 Specimen collection & transport
 Important considerations:

 Proper collection
 Rapid delivery to the laboratory
 Prompt and correct processing
 Inoculation into proper and appropriate
medium
 Incubation at a suitable temperature
Specimen collection & transport

 Transport of Specimen:

 Antibiotics may be incorporated in body fluid

specimens to prevent proliferation of bacteria:

 50, 000 units of Penicillin

 100,000 units of Streptomycin

 0.2 mg of Chloramphenicol
Specimen collection & transport

 Transport of Specimen:

 Storage temperature of specimen for fungal

culture:

 Blood & CSF: 30 – 37 OC

 Dermatological: 15 – 30 OC

 Others: 4 OC
Specimen collection & transport

 SPUTUM
 first early morning sample
 Deep cough specimen; may be induced by:
 Aqueous aerosol
 Bronchial tap
 Volume: 5 – 10 ml
Specimen collection & transport

 BLOOD and BONE MARROW

 Transport medium: at 1:10 proportion

 TSB or TSA (biphasic agar or broth)

 BHI transport medium

 Thioglycollate broth

 Volume: 10 ml
Specimen collection & transport

 CEREBROSPINAL:

 Transport immediately. Do NOT refrigerate.

 For suspected Cryptococcus, Coccidioides

infections, containers must be leak proof and
lab manipulations should be done under a hood
Specimen collection & transport

DERMATOLOGICAL SPECIMENS
SKIN LESIONS

 Sterilized area with 70% alcohol

or sterile water
 Collect at the the active border
 Specimen collection & transport
NAILS

Clean with 70% alcohol

If:
 Dorsal plate:

scrape the deeper portion

 Nail plate:

scrape beneath the nail plate

 Whole nail or clippings

Specimen collection & transport

HAIR

 Collect from:
 Areas of scaling
 Alopecia
 Hair that fluoresce under
Wood’s lamp
 Specimen collection & transport

 EXUDATES & PUS

 Undrained or unruptured abscess

 Aspirate using sterile syringe,

recap needle and transport to
lab immediately

 Failed aspiration, do skin biopsy

Specimen collection & transport

 URINE
 First early morning
 Transport and perform test ASAP
within 2 hours
 If not possible, refrigerate specimen.
 Specimen collection & transport

 VAGINAL SECRETIONS

 Sterile swabs
 Put in transport medium or
primary isolation broth
immediately (ex: TSB)
Specimen collection & transport

 TISSUES & Biopsy specimens:

 Collect aseptically at the center and edge of the

lesion

 Place in between sterile gauze wet with sterile

NSS or transport medium.
 MYCOLOGY

METHODS OF IDENTIFICATION
A. DIRECT FUNGAL MICROSCOPY

 Clinical significance:
 Provide an immediate presumptive
diagnosis
 Aid in the selection of appropriate
culture media
 Aid in decision of what’s best
inoculation technique to use
 It will provide evidence of infection despite
negative culture
A. DIRECT FUNGAL
MICROSCOPY
 Macroscopic Examination (physical exam):
 Note for:
 caseous material
 Purulent exudate
 Necrotic material
 Granules
 Punch biopsies
 Layers of skin that are broken vertically
(fissures)
 Obtain specimens for microscopy and culture
fro
A. DIRECT FUNGAL
MICROSCOPY
 Preparation for Microscopic examination:
 Mince or grind hard specimens
 Centrifuge for 3-5 minutes fluid specimens
 Pulvorize nail clippings
 Volume for fluid specimens: 0.5 ml
 Assemble a wet chamber for incubation
A. DIRECT FUNGAL
MICROSCOPY
 REAGENTS used for DIRECT MICROSCOPIC STUDY
 KOH 10-20%
 Routinely used
 10% = skin and soft tissues, body fluids
 20% = nail and hard tissues
 Calcoflour white
 Green flourescense
 India ink
 “Dark field” microscopy for Cryptococcus
neoformans
A. DIRECT FUNGAL
MICROSCOPY
 STAINS for MICROSCOPIC STUDIES:
 Lactophenol Blue
 very popular for quick evaluation of fungal structures
 stains the chitin in cell walls of fungi blue
 Use for following up fungal culture growths
 Wright’s/Giemsa stain (Diff quick)
 For rapid staining of blood and bone marrow fungi (ex:
Histoplasma capsulatum)
 Modified Acid-Fast Stain
 used to differentiate the acid-fast Nocardia from other
aerobic Actinomyces
 Gram Stain
 generally fungi are gram positive
 Actinomyces and Nocardia are gram variable
A. DIRECT FUNGAL
MICROSCOPY
 STAINS for MICROSCOPIC STUDIES:
Stains for tissue mycoses:
 Periodic Acid - Schiff Stain (PAS)
 stains certain polysaccharide in the cell walls of fungi
 Fungi stain pink-red with blue nuclei.
 Gomori Methenamine Silver Stain
 silver nitrate outlines fungi in black due to the silver
precipitating on the fungi cell wall. The internal parts of
hyphae are deep rose to black, and the background is light
green.
 Gridley Stain
 Hyphae and yeast stain dark blue or rose. Tissues stain
deep blue and background is yellow.
A. DIRECT FUNGAL
MICROSCOPY
Stains for tissue mycoses …
 Fluorescent Antibody Stain
 simple, sensitive, and extremely specific method
of detecting fungi in tissues or fluids. Applications
for many different fungal organisms.
 Mayer Mucicarmine Stain
 will stain capsules of Cryptococcus neoformans
deep rose.
 Papanicolaou Stain
 good for initial differentiation of dimorphic fungi
 Works well on sputum smears also
KOH Wet Mounts
 Principle:
 KOH softens most tissues, dissolves fat droplets, bleaches
many pigments and dissolves the “cement” that holds
keratinized cells together; glycerine clears tissue debris,
thus making it easier to demonstrate presence of fungal
elements.
 Reagents:
 10 – 20 % KOH:
 KOH pellets 10 – 20 grams
 Glycerine (optional) 10 ml
 Distilled water 90 ml
KOH Wet Mounts
 Procedure:
 Place a small amount of specimen on a clean glass slide
 place 1-2 drops of KOH on the specimen and overlay a cover
slip
 Allow the preparation to stand for 10-30 minutes in a wet
chamber.
 You can gently heat preparation to hasten the action of
KOH
 Do not over heat for it may crystallize the KOH
 Examine preparation under low then high magnification.
Take note for the presence of fungal elements (hyphae
and/or spores)
INDIA INK PREPARATION
 aka: Nigrosin stain
 Principle:
 Specimen placed in a drop of India ink becomes
darkly colored because of the carbon particle in the
ink. Hyaline structures such as capsules and cell
walls will be highlighted against a dark background
of inked colored specimen creating an illusion of
darkfield microscopy.
 Reagent: 1:1 dilution of the ink
India Ink Preparation
 Procedure:
 Place a drop of the specimen (body fluid or from culture) on
a clean glass
 Put a drop of India Ink, mix and overlay a cover slip
 Examine under low power and high power with a bright
field microscope
 Result:
 India ink creates a dark background against which hyaline
fungal cell wall and capsules can se seen
 Limitation: wbc may be confused as fungi
Lactophenol Cotton Blue
 Principle:
 The morphology of fungal elements are
preserved and stained better.
 Reagents:
 Lactic acid & Phenol
 Kills the organism
 Glycerin
 Prevents easy dehydration
 Cotton blue
 Dye or stain
DIAGNOSIS OF MYCOSES by
UNSTAINED & STAINED
MICROSCOPY
 A. SKIN or DERMATOMYCOSIS

 KOH for superficial

involvement, look for:

 Spaghetti & meat balls

(lung aspirate)
 Malassezia furfur

 Pseudohyphae and
yeasts (vaginal
secretions)
 Candida species
A. SKIN or DERMATOMYCOSIS

 KOH & LPCB for superficial

involvement, look for:

 Hyaline septate hyphae

ex: Dermatophytes

 Dematiaceous septate
hyphae
ex: Tinea nigra
Alternaria
 A. SKIN or DERMATOMYCOSIS
 H & E stain for
Oral Candidiasis
on Skin biopsy of
tongue, look for:
 Pseudohyphae
 yeasts
B. DRAINING SINUS for
MYCETOMAS & ACTINOMYCOSIS

 KOH, look for

 Various colored
granules
Actinomycosis/
Nocardiosis

 GMS stain, look for

 Various granules
Mycetoma
 C. EYE SCRAPINGS & ASPIRATE for
KERATOMYCOSIS
 KOH & LPCB, look for
 Septate hyaline hyphae
 Aspergillus species
 Fusarium species

 Coenocytic hyaline hyphae

 Mucor species

 Pseudohyphae and yeasts

 Candida species
D. NASOPHARYGNEAL ASPIRATES f for
RHINOSPORIDIOSIS
 KOH, look for

 Large sporangium
with spores (lacrimal
gland aspirate)
 Rhinosporidium
species
 E. HAIR for DERMATOMYCOSES & ALOPECIA
 KOH, look for
 Endothrix spores/hyphae
 Trichophyton

 Ectothrix spores/hyphae
 Trichophyton
mentsgrophytes
E. HAIR for PIEDRA
 KOH, look for:

 Hard, brown, compact

nodules (Black piedra)
 Piedraia hortae

 Soft, off-white,
concretions/nodules
(White piedra)
 Trichosporon beigeli
F. NAILS for ONYCHOMYCOSIS
 KOH & LPCB, look for
 Septate, hyaline hyphae
 Dermatophytes
 Epidermophyton
 Trichophyton
 Microsporon
 Pseudohyphae and yeast
cells
 Candida species
G. SYSTEMIC MYCOSES
Specimens: blood, CSF, sputum, other body fluids
KOH & Mucicarmine stain
systemic involvement, look
for:
 Pseudohyphae and yeast
cells (CSF)
 Candida species

 Broad based buds

(brain and CSF)
 Blastomyces species
SYSTEMIC MYCOSES
 GMS for systemic
involvement, look for:
 Spherules/sporangia
(CSF)
Coccidioides immitis

 PAS stain for systemic

involvement, look for:
 Dematiaceous septate
hyphae (brain tissue)
SYSTEMIC MYCOSES

 H & E stain for systemic

involvement, look for:

 Endospores (CSF brain

tissue)
 Coccidioides species

 Fission/sclerotic bodies
 Chromomyces species
SYSTEMIC MYCOSES
Wright’s/Giemsa stain (Diff
quick) & LPCB for systemic
involvement, look for:
 Small, intracellular budding
yeast (CSF)
 Histoplasma species

 Small, intracellular yeast

dividing by fission (CSF)
 Penicillin species
SYSTEMIC MYCOSES

Mucicarmine stain &

India Ink for systemic
involvement, look for:
 Encapsulate yeast
(CSF)
 Cryptococcus
neoformans
 SYSTEMIC MYCOSES

 LPCB & Fluorescent Antibody

stain for systemic
involvement, look for
 Large yeast with multiple
buds called “mariner’s
wheel”
 Paracoccidioides
braziliensis
SYSTEMIC MYCOSES
 Calcoflour mounts for
systemic mycoses , look for
(flourescence)
 Pseudohyphae and
yeasts (blood)
 Candida species

 Septate, hyaline at right

degrees angle (bronchial
lavage)
 Aspergillus species
 Interpretation of Direct Microscopic Findings (Summary)

Observation Diagnostic Possibility

Yeast and pseudohyphae Candidiasis
Fine, hyaline, septate hyphae Dermatomycosis
Clear, hyaline, septate, branching Aspergillosis
hyphae
Clear, hyaline, coenocytic, branching Mucor or Phycomycosis
hyphae
Dematiaceous, septate hyphae Tinea nigra or Phaeohypomycosis
Spaghetti and meatballs (spores and Tinea versicolor (Malassezia furfur)
hyphae)
 Observation Diagnostic Possibility

Large sporangia (>300 microns) and spores Rhinosporidiosis

Granules: varied shapes, colors, shapes and sizes Mycetomas; Actinomycosis

Brown fission bodies Chromomycosis

Broad-based yeast cells with thick walls Blastomycosis

Large, thick-walled yeast with multiple small Paracoccidioidomycosis

buds (“mariner’s wheel)

Spherules and releasing endospores Coccidioidomycosis