Dr.T.V.

Rao MD
 Importance of Fungal Infections

Fungi have a great

importance in the field of
microbiology, for they affect
all living beings. There is a
myriad of infections caused
by these agents, ranging
from superficial mycoses to
diseases with high
morbidity and mortality
rates.
 Morphology helps in
Basic Identification


 Many pathogenic fungi may
be recognized grossly by
their appearance in culture.
The more definitive method
of identification, however, is
through microscopic
examination of the
structures on which
classification depends: the
spores and the spore bearing
apparatus.
Precise methods of Identification
of Moulds


 Accurate identification of
filamentous fungi is based
on the microscopic
examination of sporulation
parts of a colony since each
species has a characteristic
morphology in the
arrangement of its spores
and fruiting bodies.
 Riddel’s Initiates Slide
Culture


 In order to accurately identify
many fungi it is essential to
observe the precise
arrangement of the
conidiophores and the way in
which spores are produced
(conidial ontogeny). Riddel's
simple method of slide
culturing (Mycologia 42:265,
1950) permits fungi to be
studied virtually in situ with as
little disturbance as possible.


Basic principles of Slide

culturing in Mycology
 Slide Culture Preparations.

One plate of nutrient

agar; potato dextrose is
recommended, however,
some fastidious fungi
may require harsher
media to induce
sporulation like cornmeal
agar or Czapek dox agar.
Slide Culture Preparations.


 1. Using a sterile blade cut out an
agar block (7 x 7 mm) small
enough to fit under a coverslip.
 2. Flip the block up onto the
surface of the agar plate.
 3. Inoculate the four sides of the
agar block with spores or
mycelial fragments of the fungus
to be grown.
 4. Place a flamed coverslip
centrally upon the agar block.
 5. Incubate the plate at 26C until
growth and sporulation have
occurred.
 Slide Culture Preparations.
 6. Remove the cover slip from the


agar block.
 7. Apply a drop of 95% alcohol as a
wetting agent.
 8. Gently lower the coverslip onto
a small drop of Lacto phenol
cotton blue on a clean glass slide.
 9. The slide can be left overnight to
dry and later sealed with fingernail
polish.
 10. When sealing with nail polish
use a coat of clear polish followed
by one coat of red Coloured polish.
 Need for Slide Culture


 Most moulds will yield good results , but a few present
extra difficulties. The most problematic are those that
tend to disintegrate as soon as they are mounted. Species
of Cladosporium, Monilia, and Alternaria have spores
connected in very fragile chains that can fall apart at the
slightest movement of air. Mounts of these fungi
invariably reveal only loose spores and a network of
hyphae. To overcome this problem it is useful to set up
slide cultures
 Slide Cultures done with
Simple Techniques


 Slide cultures are made by
setting up a small Petri dish
moist chamber containing a V-
shaped piece of glass tubing
resting on several layers of
moistened filter-paper. A
sterile block of agar medium
about 1 cm square is placed on
a flame-sterilized microscope
slide and the slide is then set in
the moist chamber on the
tubing.
 Slide culture of fungi


This procedure, which
allows fungal growth
to be examined
undisturbed. The
technique is
dependent on the
fungus attaching to a
glass coverslip.
Cut a square of agar growth medium that is
small enough to fit easily on the glass slide.


Cut a square of agar growth medium that is small enough

to fit easily on the glass slide .

Cutting the Agar


Carefully transfer this aseptically to
the center of the slide.


Carefully transfer this aseptically to
the center of the slide.


Flame the inoculating wire and allow it to
cool before inserting it into the sample .


 Sampling the fungal culture

.
Only a small amount of material is required


Inoculate one edge of the agar square with
the fungus to be examined
Inoculating the agar block.


 Agar block with all
four sides
inoculated
Sterilize the coverslips by immersing
them in alcohol.
Picking up a cover-slip with forceps


 Using sterile forceps, remove and flame a
coverslip to burn off the alcohol and place it
on top of the agar block.


In slide 4 The coverslip being placed on a slide culture
Add a little sterile water to the base of the
Petri dish to ensure that the agar does not
dry out while the culture is incubating


The duration and temperature of the incubation depend
upon the species of fungus being examined
Removing the cover-slip and its growth from a slide
culture


When sufficient growth has occurred.. lose-up of
cover-slip on a clean slide, fungal growth
uppermost.


 Place the coverslip, with the fungal growth
uppermost, onto a clean slide. A drop of alcohol
being applied to the fungal growth on the cover-slip


Add a drop of 70% alcohol to the center of
the coverslip.. Drop of lacto phenol cotton
blue on the coverslip.


 Placing a stained slide culture onto a
clean slide Turn the coverslip over and
place it onto a clean slide.


Morphology of Chlamydospores


How the Chlamydospores look


 Microscopic appearance of the slide culture at room temperature
(Czapek-Dox solution agar) after 2 weeks. Conidia in a chain with
transverse and oblique septa were observed.

Microscopic appearance of the slide culture at room temperature (Czapek-Dox

solution agar) after 2 weeks (original magnification × 200). Conidia in a chain with
transverse and oblique septa were observed.
Chlamydospores of Candida
albicans


Candida albicans in its Hyphal
Form


 Ready-to-Use, Diagnostic Slide Culture
System for Identifying Fungi

 The MycoVue™ System
provides the laboratorian with
a standardized, comprehensive
method that eliminates time-
consuming preparations and
technical difficulties
encountered with the classical
slide culture technique. It
simplifies the slide culture
method by providing all the
necessary components for this
procedure in one ready-to-use
disposable unit.
MycoVue™ System


 The system comes complete
with a protective lid and a
built-in humidifying chamber.
The device is designed to fit
easily onto a microscope stage,
thereby allowing direct
viewing of the developing
fungus through the device,
thus eliminating the disruption
of the fungal colony. If desired,
the cover slip can be removed
and stained for further
evaluation or preservation.
 References


 Microbiology Teaching Support Faculty of Biological
Support University of Leeds


Created by Dr.T.V.Rao MD for ‘e’
learning resources for Medical
Microbiologists and Mycologists in
the Developing world
email
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