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Specimen Collection
LABORATORY METHODS
FOR DIAGNOSIS OF
FUNGI

Dx of fungal infection depends entirely on

( ) selection, collection, transport and
processing of clinical specimen.

RESPIRATORY TRACT SECRETION

Sputum
Induced sputum
BW, BAL
Tracheal aspiration
Antibacterial antibiotics in the battery of media to
be used.
Cyclohexemide (0.5ml) in at least one of the
culture media (prevents overgrowth of molds)

CEREBROSPINAL FLUID
Filtered through a 0.4
um membrane filter
attached to a sterile
syringe.
Filter removed and place in
CM
Examined daily

If less than 1 ml
submitted centrifuge
and 1 drop aliquots
placed on several
areas of CM
Processed promptly if not
store at RT or at 30oC
Should not contain
antibacterial or antifungal
agents.

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BLOOD
Blood culture system
Lysis centrifugation system (for
heavy incidence of dimorphic
fungi) - effective in isolation of
H.capsulatum
Temp. 30oC for 21 days before
reporting negative

Hair, Skin, and Nail scrapings

Usually submitted for dermatophyte culture
Obtained by scraping the skin with
scalpel/microscope slide
Hairs plucking with forceps
Temp. 30oC for 21 days before reporting negative
Mycosel agar with chloramphenicol and cyclohexemide

URINE

TISSUE, BM, and STERILE BF

TISSUES: Grinded/Minced in
a Stomacher (Tekmar,
Cincinatti Ohio)

Processed immediately after collection

24 hour sample unacceptable
Centrifuge and sediment cultured using loop
Use media containing antibacterial agents to
ensure recovery

Bone Marrow: Placed directly onto the surface of

appropriate CM
Sterile BF: Centrifuged before culture , 1 ml.
Place BM and Sterile BF in an Isolator tube treat as
BC
Incubation: Temp. 30oC for 21 days before reporting
negative

Expresses cytoplasmic
contents of cells
1.O ml of suspension spread
into CM
Incubation: Temp. 30oC for 21
days before reporting negative

Microscopic Methods
Wet preparations
Saline mount direct
observation of fungal
elements

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10% KOH preparation

dissolved keratin in skin, hair,
or nails
Can be added with Calcofluor
White Stain (brightening agent)
Hair is examined for
endothrix or ectothrix
infections.

Lactophenol Cotton Blue (LPCB)

Imparts a blue color to cell wall
Used in tease mount (wet preparation) or slide
culture

Giemsa or Wrights stain

Detection of intracellular H.capsulatum in blood, lymph
nodes, lung, liver, or BM

India ink or nigrosin

Identify capsule of the yeast C.neoformans in
CSF
Difficult to interpret

Hucker Modification of Gram

stain
Fungi stain gram positive, but
poorly

Germ tube
For presumptive identification of C.albicans
Uses serum for at 37oC, 1-3 hours

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Hematoxylin and Eosin (H & E)

Histochemical stains
Useful in staining fungal elements
from deeply seated tissues (tissue
biopsy)

Determine hyaline and

Dematiaceous fungi

PAS Periodic Acid Shiff

Internal details

Mayer's mucicarmine stain

Gomoris methanamine silver stain
Provides good contrast and staining for fungal
elements
Stains viable and non-viable fungi

FontanaMasson stain
Demonstration of melanin or melanin-like
substances in the lightly pigmented agents of
phaeohyphomycosis

Primary Isolation media

Brain-heart infusion agar (BHI)
Saphrophytic and pathogenic fungi
from sterile sites

BHI w/Antibiotics (CC)

pathogenic fungi, specimens
contaminated with bacteria or
saprobes fungi

BHI biphasic culture bottles

Fungi in Blood or BM

Demonstration of the mucoid

capsule of C. neoformans

Culture Media
Generally must contain nitrogen and carbon
sources, and vitamins
Glucose , Fructose and mannose, sucrose (table
sugar)
Peptone, yeast extract, malt extract, amino acids,
ammonium and NO2 compounds.
Salts (Fe, Zn, Mn) for defined media
Thiamin (B1) and biotin (B12)

Dermatophyte test
medium(DTM)
Dermatophytes from keratinized
specimens
Can replace SDA-CC for recovery of
dermatophytes

Mycosel/Mycobiotic similar to
TDM

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Sabouraud Dextrose Agar (SDA)

Saprobic and pathogenic fungi

SDA CC

Potato Dextrose Agar (PDA)

Grows a wide range of fungi

Pathogenic fungi
Bacteria saprobe fungi inhibited

SDA with oil

For isolation of Malassezia

Cottonseed agar

Differential media
Birdseed (niger agar)

Mold phase yeast phase of

Blastomyces dermatitidis

C.neoformans, producing
phenol oxidase
resulting to production of
melanin (brown-black color)

Cornmeal agar with tween 80

Chlamydospore production of
Candida species

Czapek agar and MALT agar

Nitrate reduction medium

Confirm N03 reduction of
C.neoformans

Malt Agar (MA)

Aspergillus species
Useful in isolation of Ascomycota

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Differential test media

Urea agar
Urease production

Yeast assimilation media (carbon or nitrogen)

CHO assimilation

Yeast fermentation broth

Fermentation reaction with various CHO

Slide Culture
Optimal examination method for
preservation of fungal morphology

Biochemical tests
CHO assimilation test for yeast
Rapid urease test (UREA R broth)
Yeast fermentation tests