Laboratory Diagnosis of Fungal Infections: by Prof. Suzan El-Fiky

Laboratory Diagnosis of
Fungal Infections
By
Prof. Suzan El-fiky
INTRODUCTION
1. A presumptive clinical diagnosis of

mycosis must be confirmed by a
laboratory diagnosis.
2. Definite diagnosis is usually reached or

attained by direct culture from
pathological materials.
3.It is essential in reaching a “proved
diagnosis” to isolate the fungus in
pure culture for morphologic
examination.
4.Mycologist should not be asked to
make a mycological identification of
the fungus without being given an
opportunity to study
histopathologic sections as well as
cultures.
5.Serological diagnosis sometimes
can be helpful, but unreliable due
to cross-reactions.
6. identification of a fungus may be

delayed depending upon
experimental infection of animals
and preparation of sections of their
tissue.
Laboratory Diagnosis
1. Mycological Diagnosis
a. Sampling
b. Direct microscopical examination.
c. Cluture.
d. Identification.
2. Histopathological Diagnosis
Special fungal stains i.e.
PAS and GMS.
3. Serological Diagnosis.
DD, ELISA, IF,IHA,..etc
4. Laboratory Animals.
Sampling
1.In superficial mycoses the scales or infected
hairs may be stored into small sterile glass
Petri dishes.
2.Infected nail or skin scrapings are taken from

the deeper parts with a blunt scalpel.
3.Specimens from the mucous membranes or

from orifices should be collected with dry
swabs or preferably swabs soaked in
Sabouraud’s broth.
4. For the diagnosis of bronchopulmonary infection
morning sputum should be collected in a sterile
container.
5. For systemic mycosis, pus swab from an ulcer or

aspiration from unruptured abscess, or biopsy during
surgical operation are collected by strict aseptic
technique.
6. For urinary tract infection, mid-stream urine samples

are collected into a wide mouth sterile container.
7. For cerebrospinal infections, a
lumbar puncture should de
performed to collect CSF into sterile
test tubes.
8. For Pleural and Peritoneal Effusions,

a sample is collected by needle
aspiration into sterile container.
Sampling using blunt scalpel
Specimen Collection
Sampling using cotton swab
Sampling using Biopsy
Direct Microscopical Examination
. 1.Fragments of skin, hair, nail or drop of

pus or sputum are placed in a drop of
10-30% KOH solution on a glass slide
and a cover-slip is applied .
2. first to examine the specimen widely

with a low power objective and then to
search suspicious areas with a high
power.
3.Mycelium chains of arthrospores and
free arthrospores may be seen and
their recognition permits a diagnosis
of fungal infection. Budding yeast
cells mixed with long filaments are
indicative of a yeast-like fungus.
4-if there is doubt as to the nature of any
fungus-like elements in the specimen,
KOH is replaced by lactophenol blue, or
clacflour stain.
5. In case of Tinea versicolour use
Scotch-tape method to identify
spores.
6. Use India Ink to demonstrate the
capsule of cryptococcus.
7. Examination of air-dried, heat-fixed

or stained films is not as generally
useful in medical mycology as in
bacteriology
Direct Examination
Culture
1.The agar medium most commenly used is:
Sabouraud’s Dextrose Agar (SDA)
which has the following composition:
Dextrose 20 g
Peptone 10 g
Agar 20 g
H2O 1L
Autoclave at 121 C for 10 minutes (pH 5.4)
2. Sabouarud’s Dextrose Agar is the
most suitable medium because
fungal growth is favoured by a high
sugar concentration and is relatively
tolerant to acidity (pH 5.4)
3. The agar is prepared as slopes in

test tubes stoppered with cotton-
wool as most of the fungi are aerobic
4. If the specimens are contaminated
e.g. sputum, incorporate antibiotics
such as chloramphenicol (0.5 g/l) in
the media used for isolation.
5.Cycloheximide (Actidione*) can

also be incorporated for the
isolation of dermatophytes in order
to get rid of saprophytic fungi
6. Petri-dishes should not be used for
isolation of pathogenic fungi; the
possibility of having a confusing
contaminant is increased. Also the
use of petri-dishes is hazardous if
Dimorphic fungi are present .
7. The pathological material is spread upon
the surface of agar slopes. The fragments
of skin, hair or nail are planted with a firm
straight pointed wire. Incubate slopes at
temperature up to 30oC
Identification
When the fungus is isolated in

pure culture, examine it
macroscopically and
microscopically
Macroscopic Examination:
The texture and colour of the colony are

examined in its upper and lower sides,
i.e. Recto and Verso, the presence of
diffusible pigments in the medium
should be noted. The type of colony is
observed, yeast-like or filamentous.
Recto / Verso
Recto Verso
Microscopic Examination:
A “Needle Mount” is made as soon as spore

formation is sufficiently advanced at the
center of the colony. Place a drop of
lactophenol cotton blue on the glass surface.
With a nickel-chrome needle, pick-up a bit of
the mycelium and put it directly to the drop of
stain. Cover it with a cover-slip and examine
under the microscope.
Lactophenol Cotton Blue
Antifungal susceptibility tests
Recently antifungal antibiogram was
developed especially for fungi in yeast
forms.
Disc diffusion method was applied as well
as the micro dilution methods for testing
antifungal drugs.
Histopathological Diagnosis
To establish the diagnosis of a fungus

disease, identification should be made by
a combination of mycologic and
histopathologic study.
Although many fungi can be seen with
haematoxylin and eosin stain (H&E),
some are not stained by this method.
Special stains for fungi are of great
help in reaching the etiologic agent.
These stains are mainly:
Periodic acid-Schiff stain (PAS).
Gomori methenamine Silver stain (GMS).
Mayer’s mucicarmine stain.
Gridley fungal stain.
PAS
GMS
H&E
Mucicarmine
Serological Diagnosis
Serological diagnosis of mycoses usually
lacks complete specificity because some
of the pathogenic fungi have common
antigens.
Fractional separation of the active antigenic
components of a fungus has not been
achieved with complete success.
However, serologic techniques are useful
in reaching a presumptive diagnosis.
These methods may include:
Double Diffusion method.
Counter-immuno
electrophoresis.
Latex agglutination.
Indirect Immunofluorescence.
Indirect haemagglutination.
ELISA.
Double Diffusion Method
Counter-immuno-electrophoresis
Latex Agglutination
Indirect Immunofluorecence
Indirect Haemagglutination
ELISA
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Laboratory Diagnosis of

1. A presumptive clinical diagnosis of

2. Definite diagnosis is usually reached or

6. identification of a fungus may be

2.Infected nail or skin scrapings are taken from

3.Specimens from the mucous membranes or

5. For systemic mycosis, pus swab from an ulcer or

6. For urinary tract infection, mid-stream urine samples

8. For Pleural and Peritoneal Effusions,

. 1.Fragments of skin, hair, nail or drop of

2. first to examine the specimen widely

7. Examination of air-dried, heat-fixed

3. The agar is prepared as slopes in

5.Cycloheximide (Actidione*) can

When the fungus is isolated in

The texture and colour of the colony are

A “Needle Mount” is made as soon as spore

To establish the diagnosis of a fungus

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