DIAGNOSTIC: MYCOLOGY

Patrick Jumar S. Buenaflor, RMT

Emilio Aguinaldo College Cavite
School of Medical Technology

Transport and storage of specimen

treat all specimen potentially hazardous
rapid transport
delayed
- refrigerate 4C
- not frozen
- tissue, add sterile NSS
refrigeration is not an excuse to delay
processing

Transport and storage of specimen

aseptic condition
right side
right specimen
sterile containers
double
leak proof

General Recommendations

Use agar slants

Disinfect all bench tops daily
Do not keep potted plants
Do not allow dust to accumulate
Examine fungal culture in a hood
Use biosafety hood
Autoclave all cultures before discarding

Fungal Identification
Direct Examination
Culture
Macroscopic Examination
Microscopic Examination

Biochemical Test

Direct Examination
Microscopic examination of fungi in clinical
specimen
Wet mount with or w/o dye
KOH w/ or w/o Parker Super Quink blue-black
ink

KOH (with or w/o ink)

Protein, fats and polysaccharides are
solubilized
Tissue clears
Fungal cell wall alkali resistant due to
glucans and chitins
Gentle heating and warming hastens reaction

KOH w/ visible hyphae

KOH wet mount hair

Calcofluor white
Optical brightener dye used as whitening
agent in textile and paper industry
Binds cellulose and chitin
Fluoresce when exposed to UV and short
wavelength visible light
Uses fluorescent microscope

Calcofluor white

Alcian Blue
Detects C. neoformans in
CSF

Acid Fast
Detection of
Mycobacteria and
Nocardia

Giemsa
Examination of bone
marrow and blood smear

Gram Stain
detection of
bacteria

India Ink
Detection of C.
neoformans in CSF

Methylene Blue
Detection of fungi
in skin scrapings

Methenamine Silver
detection of fungi in histologic sections

Papanicolaou
examination of secretions
presence of malignant cells

Periodic Acid Schiff

detection of fungi
histochemical stain for glycogen

Wrights Stain
Examination of bone marrow or PBS

Problems with Direct Examination

Negative results never rules out a fungal
infection
False + findings occur as:
- fat droplets as yeast
- collagen fibers as Nocardia filaments
Equivocal results must be reviewed by
more than one reader
Cultures should be performed on clinical
specimen

Fungal Culture
Sufficient amount
1-2 ml to inoculate 3-4 media
concentration of fluids by centrifugation
or filtration
Temperature:
RT 25C
BT37C
To encourage dimorphic fungi to produce yeast
form

Time of Culturing Fungi

Dermatophytes:
at least 2 weeks

Opportunists:
less than a week

Systemic:
4-6 weeks; if H. capsulatum is suspected up to
12 weeks

Prevent dehydration:
thick volume of agar, inside plastic bag

Observe every 2-3 days for growth wait up

to full term of incubation

Fungal Culture Media

Primary recovery media
Differential test media

Primary recovery media

BHIA
BHI with antibiotics
BHI blood culture
Inhibitory mold agar
Mycosel or mycobiotic agar
SABHI
Potato flake agar
Yeast extract PO4 agar

Primary recovery media

Brain Heart Infusion Agar
Primary recovery of saprobic and pathogenic
fungi
BHI with antibiotics
Recovery of fungi exclusive of dermatophytes
BHI biphasic in b.c. bottle
Recovery of fungi from blood

BHI for Blood Culture

SABHI

BHIA

Dermatophyte test medium

Screening medium for dermatophytes
Inhibitory Mold Agar
Recovery of fungi exclusive of dermatophytes
Potato Flake Agar
Saprobic and pathogenic fungi

Potato Flake Agar

Mycosel or Mycobiotic Agar

Primary recovery of dermatophytes
SABHI
Saprobic and pathogenic fungi
Yeast Extract PO4 Agar
Recovery of fungi exclusive of dermatophytes

Mycobiotic Agar

SABHI

Yeast extract agar

Differential Test Media

Ascopore agar
Corn meal agar
Cottonseed conversion agar
Czapeks agar
Niger seed agar
Potato dextrose agar
Rice medium
Trichophyton Agar
Urea agar
Yeast fermentation broth/Yeast nitrogen base

Differential Test Media

Ascopore Agar
Detection of Ascopores in ascoporogenous yeast
such as Saccharomyces sp.
Corn Meal Agar with Tween 80 and Tryptan blue
Identification of C. albicans by chlamydospore
production.
Identification of C. albicans microscopic morphology

Differential Test Media

Cottonseed Conversion Agar
Conversion of dimorphic fungus B.
dermatitidis from mold to yeast form
Czapeks Agar
Recovery and differential identification of
Aspergillus sp.

Czapeks Agar

Differential Test Media

Niger Seed Agar
Identification of C. neoformans
Nitrate Reduction Medium
Detection of nitrate reduction in
confirmation of Cryptococcus sp.
Potato Dextrose Agar
Demonstration of pigment production by T.
rubrum; preparation of microslide culture

PDA

Differential Test Media

Rice Medium
Identification of M. audounii
Trichophyton agars 1-7
Identification of members of Trichophyton genus
Urea Agar
Detection of Cryptococcus spp.; differentiate T.
mentagrophytes from T. rubrum;
detection of Trichosporon spp.

Rice medium

Trichophyton Agar

Differential Test Media

Yeast Fermentation Broth
Identification of yeasts by determining
fermentation
Yeast Nitrogen Base agar
Identification of yeasts by determining
carbohydrates assimilation

Identification of culture
Morphology
macroscopic
microscopic examination
Physiology (urea utilization, thiamine
requirement etc.)
Multiple Test System (API AUX System)
Biotech nuclear probes

Fungal Culture can be examined

microscopically
using lactophenol
cotton blue stain

Specimens

Abscess
Blood
CSF & other sterile body fluids
Hair
Nail clippings/shavings
Tissue
Sputum

Specimens
stool
urine (SPA, clean catch or catheterized)
vaginal secretions

Skin scrapings
clean lesion with
periphery (70% ROH)
If inflamed or w/
fissures clean with
sterile water
Use sterile scalpel or
microscope slide edge

Scrape active edge center heals first

Collect moist exudate for Candida
Place between 2 slides, envelope, petri dish
Store at room temp

Nail clippings/shavings

clean w/ 70% ROH

scrape 4-5 times, discarded
From proximal to distal
Debris under nail plate should also be
collected
Small clippings are preferable, for easier KOH
digestion

Nail clippings/shavings
10-30% KOH; overnight digestion
between 2 slides, envelope, petri dish
Store at room temp

Clean nail w/ alcohol pad

Clip toe nail as far as back consider

patients comfort

Use 1 mm curette to collect

subungual debris

Good specimen in envelope

Sputum

1st morning
after brushing & mouthwash
Wide mouth container
12-72h collection, unacceptable
4C storage/transport

Hair
Examine scalp w/ Woods lamp for fluorescing
hair
Sterile forceps, tape
Collect fluorescent hair or broken hair
Scrape scalp scales from affected area
Place between 2 slides, in an envelope or petri
dish
store RT

Tissue
collected by physician
must consist of normal tissue & both the
center and edge lesion
H. capsulatum: center
B. dermatitidis: edge
Between 2 moist sterile gauze pads, sterile
tubes or vial w/ sterile water
Not formalin

Urine
sterile containers
sent immediately
24h urine
unacceptable
From catheter bag
unacceptable

Abscess
Aspirated aseptically by physician
sterile tubes

Blood
Aseptic collection
Minimum of 5 ml
added to broth to
get 1:10 to 1:20

CSF and other body fluids

aseptic collection
lumbar puncture
process immediately
avoid refrigeration

Vaginal secretions
20% of healthy female, has yeast as normal
flora
Vaginal candidiasis better diagnosed or
established by clinical characteristics and
direct examination

Stool
40% of healthy and 75% of compromised
patients have yeasts in GIT