Fungal Isolation

Learning Activity No. 11

Introduction
 Fungi
• Eukaryotic organisms that have cell walls
and obtain food from other organisms.
• Molds, mushrooms, and yeasts are
chemoheterotrophic. Their cell walls are
composed of a strong, flexible, nitrogenous
polysaccharide called chitin.
• Fungi acquire nutrients by absorbing
dissolved organic material through their
cell walls and plasma membranes.
• Compared to bacteria, fungi generally grow
better in more acidic conditions and
tolerate higher osmotic pressure and lower
moisture.
 Yeasts
• Nonfilamentous, unicellular fungi that are
typically spherical or oval in shape.
• Found in fruits and leaves as a white
powdery coatin
• Composed of a single cell which may have
buds
• When buds fail to detach themselves, a
short chain of cells called pseudohypha
forms
• Metabolic activities are used to identify
genera (singular: Genus) of yeasts
• Yeasts are facultative anaerobes.
Metabolic activities are used in many
industrial processes.
 Molds
• Multicellular filamentous fungi

• Thallus: macroscopic mold colony

• Mycelium: composed of mass of

strands
• Aerial hyphae=reproductive hyphae
originate from the vegetative hyohae
and produce a variety of asexual
reproductive spores.
• Septate Hyphae: hyphal strand of mos
molds composed of individual cells
separated by a septum
• Coenocytic hyphae: have hyphae that
lack septa and are continuous mass of
cytoplasm with multiple nuclei.
• Sabouraud Dextrose Agar: has a slightly
low pH, which by inhibiting the growth of
bacteria is selective for fungi.
Procedures
I. Unicellular Fungi (yeast)
• 1. A pinch of baker's yeast in a small amount of lukewarm water in a test
tube.
• 2. Divide one Sabourde agar plate in half. Streak one half with known
yeast culture and the other half with the baker's yeast suspension.
• 3. Incubate all media at 35•C until growth is seen.

• 4. Observe morphology of yeast in wet mount and mix with methylene

blue.
• 5. After the yeast have grown, record your results.
II. Multicellular Fungi (molds)
• 1. Aseptically, with a pair of forceps, place a sheet of sterile filter paper in
a petri dish.
• 2. Place a sterile U-shaped glass rod on the filter paper.

• 3. Pour enough sterile water on filter paper to completely moisten it.

• 4. With forceps, place a sterule slide on the U-shaped rod.

• 5.Gently flame a scalpel to sterilize, and cut a 5mm square block of the
medium from the plate of Sabouraud's agar.
• 6. Pick up the block of agar by inserting the scalpel into one side.
Inoculate both top and bottom surfaces of the cube with spores from the
mold colony.
• 7. Place the inoculated block of agar in the center of microslide.

• 8. Aseptically, place a sterile cover glass on the upper inoculated surface

of the agar cube.
• 9. Place tge cover on the petri dish and incubate at room temperature for
48 hours.
• 10. After 48 hours examine the slide under LPO. If growth is inadequate
allow the mold to grow another 24-48 hrs before making the stained slide.
Part 2.
• 1. Place a drop of lactophenol cotton blue stain on clean microscope slide.

• 2. Remove the cover glass from the slide culture and discard the clock of
agar.
• 3. Add drop of 95% ethanol to the hypae on the cover glass. As soon as the
alcohol evaporated place the cover glass, mold side down, on the drop of
lactophenol cottom blue
• 4. Remove the slide from the petri dish, add a drop of 95% ethanol to tge
hypae and follow this up with a drop of lactophenol blue stain. Cover the
entire preperation with a clean cover glass.
• 5. Compare both stained slides under the microscope.
III. Dermatophytes
• 1. Obtain scraping from skin of foot of a subject for mi robiology study.

• 2. Seat the subject with his bare feet resting on paper towels placed on the
floor.
• 3. Examine the skin of the subject's feet for sign of fungus infection - scaling,
fissures, maceration.
• 4. Swab a suitable area of infection with 70% alcohol.

• 5. In the absence of infection, swab the skin between the fourth and fifth toes.

• 6. Prepare a wet mount of specimen skin scraping.

• 7. Inoculate the culture medium with specimen skin scrapings

IV. Fungal Population in the laboratory
• 1. Place a petri plate of Sabouraud agar near your workbench.

• 2. Remove lid for the duration of the laboratory period.

• 3. Just before you leave for the day, replace the lid.

• 4. Incubate plate at 30•C until next laboratory period.

• 5. Observe the amount and nature of fungal growth.

• 6. Study representative samples of fungal growth in wet mounts and on

stained smears.
Results
Fruit Molds
Bread Mold
Dermatophytes
Yeast
Conclusions
• Most fungi are multicellular like the mold it consists of filaments called
hyphae that can bunch together into structures called mycelia, but some,
the yeasts, are simple unicellular organisms. Fungi are recognized by
their morphology within culture that being use. Fungi have mycelium
and spores which are used in the identification. Therefore you have to
search for mycelium (hyphae), the spores, origin of the spores, asexual or
sexual; and their structure and morphology.
Learning Evaluation
1. What important structure in yeast is not
seen as a specific entity in bacteria?
• Yeasts are unicellular eukaryotic fungi with completely different
properties than those of bacteria, which are Prokaryotes; do not have a
membrane enclosed nucleus. Yeast contains almost the same organelles
of a mature eukaryotic cell. Nucleus, Golgi apparatus, mitochondria,
endoplasmic reticulum, vacuole, and cytoskeleton are the most important
one. Among yeast, S. cerevisiaeis of industrially important due to its
ability to convert sugars (i.e., glucose, maltose)into ethanol and carbon
dioxide (baking, brewing, distillery, liquid fuel industries). S.
cerevisiae breaks down glucose through aerobic respiration in presence of
oxygen. If oxygen is absent, the yeast will then go through anaerobic
fermentation. Another common use of yeast is in the rising of bread. The
carbon dioxide that is produce inside the dough causes it to rise and
expand. In the baker’s yeast, these have strains that produce dioxide are
more prevalent than ethanol and vice versa for brewing industries.
2.Make drawing of the fruit bodies of
Rhizopus and Penicillium.
3. Why are antibiotics added to the
sabouraud dextrose agar used to isolate
dermatophytes from the skin of the feet?
• Avoid the bacteria from growing. Most fungi are
slow growing, at least compared with bacteria. If
antibiotics weren't added to the agar, you'd find a
plate full of bacteria, and not the fungus. Sabouraud
Dextrose Agar (SDA) is used for the isolation,
cultivation, and maintenance of non-pathogenic and
pathogenic species of fungi and yeasts.
 4. Explain the use of 10% potassium
hydroxide as a clearing agent in the
demonstration of dermatophytes?
• Potassium hydroxide (KOH) preparation is used for the rapid
detection of fungal elements in clinical specimen, as it clears
the specimen making fungal elements more visible during
direct microscopic examination. KOH is a strong alkali.
When specimen such as skin, hair, nails or sputum is mixed
with KOH it softens, digests and clears the tissues (e.g.,
keratin present in skins) surrounding the fungi so that the
hyphae and conidia (spores) of fungi can be seen under
microscope.