MYCOLOGY

LABORATORY
DIAGNOSTIC

LAB ACTIVITY MANUAL

Purposes :

To become familiar with the

macroscopic and microscopic
structure of yeast and mold
To become familiar with basic
mycological culturing and
microscopic examination procedures
To provide an opportunity to identify
a common fungal organism
Identification of fungi base
on the morphology of
the :
• Colony or thallus
• Hyphae or mycelium
• Spores
Colony morphology :
• Filamentous colony (mold)
• Yeast colony
• Yeast like colony
Filamentous colony (mold) :
• Macros : cottony, velvetty,
fluffy, etc
• Micros : hyphae and spores
E.g. :
Aspergillus
Penicillium
Rhizopus
Filamentous colony (mold)
Hyphae and spores
Yeast colony :
• Macros : creamy
• Micros : yeast cells
E.g. :
Cryptococcus
Blastomyces dermatitidis
Yeast colony
Yeast cells
Yeast like colony :
• Macros : creamy
• Micros : yeast cells and
pseudohyphae
E.g. : Candida sp.
Yeast like colony
Candida sp.
Hyphae (mycelium) :
• Septate hyphae, e.g. :
Aspergillus
Penicillium
Phialophora
Septate hyphae
Hyphae (mycelium) :
• Non-septate hyphae
(coenocytic)
E.g. :
Mucor
Rhizopus
Absidia
Non- septate hyphae
The spores :
• Asexual spores
• Sexual spores
Asexual spores
• Blastospore (blastoconidia)
• Arthrospore
(arthroconidia)
• Chlamydospore
• Sporangiospore
• Conidiospore (conidia)
Blastospore :
• Produce by the yeast cells as
budding
E.g. :
Candida albicans
Cryptococcus neoformans
Blastomyces dermatitidis
Blastospore
Arthrospore :
• Produce by fragmented
hyphae
E.g. :
Coccidioides immitis
Geotrichum candidum
Dermatophyte fungus
Arthrospore
Chlamydospore :
• Produce by hyphae
• The hyphae became round
and has a thick wall
E.g. : Candida albicans
Chlamydospore
Sporangiospore :
• The spores that produce in
a sac called sporangium
E.g. :
Rhizopus
Mucor
Absidia
Sporangiospore
Conidiospore (conidia) :
• The spores that protrude
from specific hyphae
called conidiphore

Microconidia : small,
unicelluler
Macroconidia : large,
multicelluler
Penicillium
Aspergillus
Microsporum canis
Epidermophyton floccosum
Sexual spore :
• Ascospore
• Basidiospore
• Zygospore
Ascospore
Basidiospore
Zygospore
Mycology Laboratory
Examination

Macroscopic examination :
Material :
7 – 10 days Sabouraud agar culture
of molds and yeast colony
Procedures :
Examine both of the colony with
magnifying glass
Describe characteristic of each
colony
Microscopic examination :
- is an essential step in
diagnostic of mycosis
- Provide a rapid and tentative
diagnostic
• Material :
– 7 – 10 days Sabouraud agar
culture of Aspergillus sp. and
Candida sp.
• Equipment :
– Spiritus burner, glass slides,
coverslips, inoculating loop
and needle, KOH sol of 10% (or
lactophenol cotton blue)
• Procedures :
– Using the loop, add 2 loopfulls
of KOH 10% sol to a clean glass
slide
– With a sterile loop, touch the
loop to the culture and
emulsify the cells in the drop
of KOH 10% sol
– Place a coverslip on the
emulsifying drops
– Examine each mycological
slides preparation under low
Culture :
• Material :
– 7 – 10 days Sabouraud agar
culture of molds and yeast
• Procedures :
– With a sterile inoculating loop,
inoculate appropriately
labelled Sabouraud Dextrose
Agar plate with one of each
colony
– Incubate at room temperature
(25OC – 27OC) for one to three
weeks.
Note :
• Succesfull isolation of a
fungus causing disease in
dependent upon each of the
following factors :
– Proper collection of specimen
– Proper handling and transport
of the specimens
– Prompt and correct processing
of the specimens
– The expertise of the
technologist for identifying the
fungus
Specimens should be
processed as soon as
possible :
• To ensure that the infecting
fungus does not die
• To control contaminating
organisms
The specimens could be :
• Skin scraping, nail scraping,
hair, biopsied tissue, exudate
or pus, sputum, blood, spinal
fluid and urine
Culture media for isolation and
identification :
The isolation medium
selection depend
upon :
• The type of specimen
(heavily contamined or
sterils)
• The suspected etiological
agent
A non selective medium like
Sabouraud Dextrose Agar
(SDA) should be routinely
use because it will support
the growth of almost the
medically important fungi
Temperature of incubation :
• Is important in the primary
isolation of fungi
• May be room temperature
(25OC – 27OC)
• May be at 37OC
• Can act as selective factor
(incubating at 45OC will
inhibit most fungi and
bacteria, but not Aspergillus
fumigatus)
 Slide cultures
technique

A block of sterile agar is cut out of a Petri dish (A) placed upon a sterile slide
resting on a bent glass tube within a sterile Petri dish (B) A few spores of a
fungus are inoculated at the edges of the sterile agar block (C) topped with a
cover-glass (D) for incubation A disc of moist filter-paper in the dish maintains
humidity for the culture
 Microslide Culture

1. Cut a small block of a suitable agar medium that has been

previously poured into a culture dish to a depth of approximately 2
mm. The block may be cut using a sterile scalpel blade or with a
sterile test tube that has no lip (which produces a round block).
2. Place a sterile microscope slide on the surface of a culture dish
containing sterile 2% agar. Alternatively, place a round piece of
filter paper or paper towel in a sterile culture dish, add two
applicator sticks, and position the microscope slide on top.
3. Add the agar block to the surface of the sterile microscope
slide.
4. With a right-angle wire, inoculate the four quadrants of the
agar plug with the organism
5. Apply a sterile coverslip to the surface of the agar plug.
6. If the filter paper applicator stick method is used, add a
small amount of sterile water to the bottom of the culture dish.
Replace the lid of the culture and allow it to incubate at 30°C.
7. After a suitable incubation period (and working inside a biologic
safety cabinet), remove the coverslip and place it on a microscope
slide containing a drop of lactophenol cotton or aniline blue. Some
suggest placing the coverslip near the opening of an incinerator-
burner to allow the organism to dry rapidly on the coverslip before
adding it to the stain.
8. Observe microscopically for the characteristic shape and
arrangement of spores.
9. If the microslide culture is unsatisfactory for microscopic
identification, the remaining agar block may be used later if it is
allowed to incubate further. The agar plug is then removed and
discarded, and a drop of lactophenol cotton or aniline blue is placed
on the area of growth and a coverslip is positioned in place.
Germ Tube Test

1. Suspend a very small inoculum of yeast cells obtained

from an isolated colony in 0.5 mL of sheep serum or rabbit
plasma.
2. Incubate the tubes at 35° to 37°C for no longer than 3
hours.
3. After incubation, remove a drop of the suspension and
place on a microscope slide. Examine under low-power
magnification for germ tubes.
 A germ tube is an appendage that is half the width and
three to four times the length of the yeast cell from which it
arises.
 No point of constriction exists at the origin of the germ tube
from the cell.
Terima Kasih