Laboratory Diagnosis

Safety Issues
Specimen Collection,
Handling & Transport
Microscopic Methods
Isolation Methods
 Safety Issues
• Class II BSC: to reduce personnel
exposure to fungal elements.
-must be checked: ____________
• Enclosed electric incinerator:
recommended to eliminate the hazards
of open gas flames and contain particles
emitted when loops or needles are
incinerated
• Screw-top tubes are recommended
Safety Issues
Laboratory Diagnosis

Safety Issues
Specimen Collection,
Handling & Transport
Microscopic Methods
Isolation Methods
 Specimen Collection,
Handling & Transport
General guidelines:

-All specimens for mycology should be transported and processed as soon as

possible.
-Anaerobic transport media or anaerobic containers should never be used for
fungi.
-Specimen containing normal bacterial flora must be transported immediately.
-Must be processed immediately or as soon as possible.
-Unlabeled specimens are rejected.
 Specimen Collection,
Handling & Transport
Collection of appropriate specimens: the primary
criterion for accurate diagnosis of mycotic infections

Most common specimens:

1. Respiratory Secretions Opportunistic + Dimorphic
2. Hair
3. Skin Superficial + Dimorphic
4. Nails
5. Tissue Subcutaneous + Dimorphic
6. Blood
7. Bone Marrow Systemic
8. CSF
THROAT, URINE, VAGINAL, CERVICAL: Candida
Specimen Collection,
Handling & Transport
 Respiratory Specimens
- Sputa from a deep cough shortly after arising in the morning.
- Collect into a sterile, screwtop container.

- Not too viscous: specimens can be inoculated onto media with a

sterile pipette
- Medium with antimicrobial agents: prevent bacterial overgrowth
- A KOH preparation should also be made.

- Nasal sinus specimens obtained surgically: plated directly to

media containing antimicrobial agents except for cycloheximide
Specimen Collection,
Handling & Transport
 Blood and Bone Marrow
Lysis centrifugation system: most sensitive method
for the recovery of some fungi

Biphasic system (broth and agar) can also be used.

Heparinized bone marrow specimens should be

plated directly onto media at bedside; Blood
culture bottles are not recommended.
Blood and Bone Marrow
Specimen Collection,
Handling & Transport
 Abscess Fluid, Wound
Exudates and Tissue

Use a dissecting microscope.

Tissue should be gently minced

before inoculation.
Specimen Collection,
Handling & Transport
SKIN

1. Skin samples are scraped from the

outer edge of a surface lesion.
2. Skin must be cleaned with 70%
isopropyl alcohol before sampling.
3. KOH wet mount is prepared.
4. Remaining material is inoculated
directly onto the agar.
 HAIR
Wood’s lamp
- emits ultraviolet light > 365 nm
- useful in identifying infected hairs

Sterile forceps
- used to pull affected hair

Less useful method

1. cutting the hairs close to the scalp with sterile scissors
2. placed directly into a sterile Petri dish
3. few pieces of hair are inoculated onto fungal medium and
incubated at 22° C to 30° C
 NAILS

- may be submitted as scrapings or cuttings and

occasionally as a complete nail
- cleaned with 70% isopropyl alcohol before the
surface is scraped
- deeper scrapings: necessary to prepare a KOH
preparation and inoculate media
- sterile scissors: cut complete nails into small thin
strips
 Specimen Collection,
Handling & Transport

.
 Cerebrospinal Fluid

Should be concentrated by centrifugation before

inoculation.

One drop of the concentrate is used for India ink

preparation or latex agglutination for
Cryptococcus, and the remainder is inoculated
onto media.
 Urogenital Specimens

Should be centrifuged and the sediment will

be used to inoculate media.

A first-voided morning urine specimen is

preferred.
Laboratory Diagnosis

Safety Issues
Specimen Collection,
Handling & Transport
Microscopic Methods
Isolation Methods
 Microscopic Methods

Provides a rapid report to the physician.

Specific morphologic characteristics provide a clue to the genus of the organism.

Might provide evidence of infection despite negative cultures.

 KOH Preparation

10% to 20% solution KOH: useful for detecting fungal

elements embedded within skin, hair, nails, and tissue.
1. drop of the KOH preparation is added to a slide
2. nail scrapings, hair, skin scales, or thin slices of tissue are
added to the drop
3. cover slip is added
4. slide is then gently heated and allowed to cool for
approximately 15 minutes
 KOH Preparation

Modifications of the KOH test: provide easier and

more reliable results
1. incorporate dimethyl sulfoxide (DMSO)
2. stain into the KOH solution
 KOH with Calcofluor White

Calcofluor White
- binds to polysaccharides present in
the chitin of the fungus or to cellulose
- fungal elements fluoresce apple green
or blue-white
 India Ink (Nigrosin)

- Negative stain that is used to examine CSF for the

presence of the encapsulated yeast C. neoformans

1. drop of India ink + drop of sediment from a

centrifuged CSF specimen

2. examined on high magnification (×400)

- budding yeast surrounded by a large clear area
against a black background is presumptive evidence of
C. neoformans
 Tissue Stains
Common tissue stains
1. Periodic Acid–Schiff (PAS): attaches to
polysaccharides in the fungal wall — stains fungi
pink
2. Gomori methenamine–silver nitrate
3. Hematoxylin and eosin
4. Giemsa: used primarily to detect H. capsulatum
in blood or bone marrow
5. Fontana-Masson: stains melanin in the cell wall
and identifies the presence of phaeoid fungi
 Tissue Stains
Common tissue stains
1. Periodic Acid–Schiff (PAS): attaches to
polysaccharides in the fungal wall — stains fungi
pink
2. Gomori methenamine–silver nitrate
3. Hematoxylin and eosin
4. Giemsa: used primarily to detect H. capsulatum
in blood or bone marrow
5. Fontana-Masson: stains melanin in the cell wall
and identifies the presence of phaeoid fungi
 Germ Tube

- most important and easiest test to perform

for the identification of yeasts
1. requires the use of serum or plasma, such
as fetal bovine serum
2. substrate is inoculated
3. incubated at 37° C for 3 hours
 Germ Tube
- presumptive identification of C.
albicans or C. dubliniensis
1. True germ tubes lack constriction
at their bases, where they attach to
the mother cell.
2. C. dubliniensis is differentiated from
C. albicans by its inability to grow at
42° C.
Laboratory Diagnosis

Safety Issues
Specimen Collection,
Handling & Transport
Microscopic Methods
Isolation Methods
 Isolation Methods
CULTURE MEDIA
• For Primary Isolation: Sabouraud dextrose agar (SDA), Potato
Dextrose Agar (PDA) and BHI agar enriched with blood and
antimicrobial agents
• Antimicrobial agents: Gentamicin or chloramphenicol and
cycloheximide

1. Petri dishes: larger surface area, but are more prone to

dehydration; must be poured thicker than a standard medium for
bacterial growth
2. Tubed media: safer to handle and less susceptible to drying
 Isolation Methods

Incubation Information that should be recorded

• Routine: room temperature or at 30° C • number of days until first visible growth
• Dimorphic fungus: cultures should also • number of days required to see fruiting
be incubated at 37° C structures
• Maintained for 4 to 6 weeks and should • media on which the fungus is isolated
be examined twice weekly for growth • temperature at which growth occurs
• morphology of the colonies
 Isolation Methods
Differential Media
A. Birdseed (Niger Seed Agar) – C. neoformans (black-brown colonies).
- Detects phenol oxidase
B. Cornmeal Agar w/ Tween 80 – Candida spp.
- Stimulates conidia & chlamydospore production.
C. Cottonseed Agar – Blastomyces dermatitidis
- Stimulates conidia production
D. Potato Dextrose Agar – Induces pigment production of Trichophyton rubrum
- Stimulates conidia production
E. Rice Medium – Differentiation of Microscporum spp,
 Isolation Methods
Differential Media
F. Urea Agar – Differentiation of Trichophyton rubrum & Trichophyton mentagrophytes

G. Yeat Test System

1. Yeast assimilation media – utilization of carbon and nitrogen
2. Yeast fermentation broth – fermentation of carbohydrates