MICROBIOLOGY PRAX REVIEWER:
Two advantages of KOH preparation:
Exercise 10: KOH MOUNT
KOH (potassium hydroxide) Mount
- is a rapid method for the demonstration of
fungal forms in clinical material
- facilitates the clearing of specimen for
enhanced microscopic observation without
altering the fungal elements.
- when a specimen if placed in KOH, the
specimen will dissolve faster than the fungi
since the chitinous cell walls of the fungi will
protect it from disintegration
A. Collection of Specimen
1. Clean the area with 70% alcohol and
allow to air dry
2. With a sharp scalpel, scrape the
epidermal scales at the active edge
of the skin or nail lesion.
B. Microscopic Examination
1. Place specimen on the glass slide
2. Add 1 – 2 drops of 10% KOH for
skin scrapings and 20% KOH for
nails and hair specimen
3. Put a cover slip and let the mount
set for 10 – 30mins. If material is
hard (nail or tissue), heat the slide
gently by passing it over the flame
once of twice. Do not overheat or
KOH will crystallize.
4. Examine the mount under the
microscope. Note the presence of
granules, hypal elements, spores or
yeast cells.
Demo Slide:
1. Facilitates clearing of specimens for enhanced
microscopic observation without altering
fungal elements
2. Simple, cheap and rapid
Two disadvantages of KOH preparation:
1. Gives an idea about the presence of hypal
elements, but cannot distinguish different
fungi
2. Preparation cannot be kept for too long
Three factors that may affect the results of a
KOH preparation:
1. Pressure application
2. KOH droplet size
3. KOH indication
Exercise 11:
Microscopic Morphology of Fungal Culture
 Identification of fungi is often accomplished
by isolation organisms on appropriate culture
media and observing their morphological
characters
 Accurate Identification of filamentous fungi is
based on the microscopic examination of
sporulating parts of a colony, since each
specie has a characteristic morphology and
arrangement of its spores and fruiting bodies
Laboratory Techniques:
Tease Mount Preparation
- technique traditionally used by most
laboratories
- primary purpose is to demonstrate conidia
or other reproductive structures or
morphological forms which might give
information toward the identification of the
organism
Procedure:
1. Using an inoculating needle, pick a small
portion of each fungus growth and place on
separate slides containing a drop of LPCB
(Lacto-phenol cotton blue)
2. Tease with inoculating needle. Emulsify the
growth of the yeast and yeast-like fungi
3. Put a cover slip and examine under low and
high power objectives.
4. Observe record and draw the following
important morphological features of the
various fungi. E. Floccosum
a. Nature and type of mycelium
(presence or absence of crosswalls)
b. Characteristic arrangement, size,
shape, and type of spores produced
(blastospores, arthrospores,
chlamydospores, sporangiospores,
macro/microconidia, etc) and some
special characteristic
c. Nature of growth (fluffy, velvety,
powdery, dry, moist)
d. Color of growth on surface and
reverse side.
5. Label all drawings accordingly.
6. Observe and characterize the different fungal
colonies

Demo Slides:

I. Trichophyton Mentagrophyte Candida Albicans

Microsporum Gypseum

Slide Culture or van Tieghem Cell

- Considered the best method for preserving
and observing the actual structure of a
fungus.
- in an undisturbed state, important
microscopic structures and morphologic
details are demonstrated

Procedure:
1. Inoculate the microculture plates separately
with each of the given fungus.
2. Hold a microscope coverslip with a pair of
forceps and put it on top of the agar inside the
plates.
3. Check the culture periodically for growth and
sporulation. When the fungus has grown onto
the square of the Sabouraud’s dextrose agar
and out onto the small part of the slide and
coverslip, proceed with the next step.
Incubation time varies with each fungus,
usually 5 – 10 days for contaminants and 1 – 3
weeks for pathogens.
4. Remove the slide from the petri dish. Take a
new clean slide, and place a drop of LPCB on it
near the center and put the coverslip that has
fungus growth from the other slide. Label the
slide.
5. Examine the slide for reproductive structures
and notice the undisturbed morphology.

Demo Slides:

Penicillium sp.
Aspergillus sp
a. Small, ovoid structures dividing by transverse fission
b. Flat, powdery to velvety, tan to reddish yellow colonies a. Mycelia
c. Conidiosphore bearing short broad metullae b. Fruiting heads
d. At 37C colonies are soft and yeast-like, round or ovoid c. Septate, monomorphic fungi
cells with central septum are seen d. Forms branches at acute angles
 Disadvantages of slide Culture:

Waiting period for incubation of slide culture

Advantages of Tease Mount Preparation:

This can be done easily and quickly

Disadvantages of Tease Mount Preparation:

The characteristic arrangement of conidia may be

disrupted when pressure is applied to coverslip.

Advantages of Scotch Tape Lactophenol Mount:

Easy and fast procedure that is used for

identification of filamentous fungi since most
structures will be intact for observation.

Disadvantages of Scotch Tape Lactophenol

Mount:

Tape will dissolve eventually so that it is not used

for permanent mounts, the procedure can only be
performed on moulds growing on plates.

Lactophenol Cotton Blue (LPCB)

- is very popular for quick evaluation of fungal
structures
- 3 components:
lactic acid which preserves the fungal
structures
cotton blue which stains the chitin present
in the cell walls of fungi
phenol which kills any live organisms
suspended in the stain

Advantages of Slide Culture:

Fungal elements are grown and maintained in

their original juxtaposition, thus making it easier
to morphologically identify the organism

2 mounts obtained from one culture

Simple and relatively inexpensive