FUNGI

- eukaryotic
- spore-bearing, heterotrophic organisms
- exhibit sexual/asexual reproduction
- has rigid cell (chitinous/cellulosic)
- made up of filamentous somatic structures
- has ergosterol in their cell membrane

MYCOLOGY
- study of fungi (“mykes” = mushroom)

YEAST
- unicellular, mostly reproduce by budding
- some would form buds that would fail to detach from its mother cell forming a pseudohyphae

***Candida albicans - Sabouraud dextrose agar (SDA)

MOLDS
- multicellular
- Produces Hyphae - fundamental tube-like or filamentous structures
- Mycelium/Mycelia - mat/mass of hypha that makes up the thallus of the fungi
(can be found in soil)

● Vegetative hyphae: Dense, close to the surface.

● Aerial hyphae: Fuzzy, extending upwards.

DIMORPHIC FUNGI
Exhibits both unicellular (yeast) and multicellular (mold) structures

Triggers: Factors like temperature, oxygen levels, and nutrient availability induce this switch.

FUNGAL REPRODUCTION
Sexual Reproduction: involves meiosis
Asexual reproduction: involves mitosis

Sexual spores are produced by the teleomorph form of a fungus.

Asexual spores are produced by the anamorph form of a fungus.

ASEXUAL SPORES:
(1) Conidia - originates from phialides or transforms from vegetative cells (present in most
pathogenic fungi) Arthrospores, Blastoconidia and Chlamydospores are also conidia
(2) Sporangiospores - produced in a structure called sporangium (found in Order
Mucorales)
FUNGAL CLASSIFICATION

Zygomycetes
● Mycelium: Non-septate.
● Asexual spores: sporangium; sporangiospores (nonmotile).
● Sexual spores: Zygospores (motile), found in terrestrial forms. Oospores, found in
aquatic forms.
● Common species: Bread molds, mildews, potato blight (Rhizopus, Mucor).

Ascomycetes
● Mycelium: Septate.
● Asexual spores: conidiophores; conidia (nonmotile).
● Sexual spores: Ascospores, contained in a sac-like structure called the ascus.
● Common species: Cup fungi, ergot, Dutch elm disease, yeast species (Peziza, Pichia,
Saccharomyces).

Basidiomycetes
● Mycelium: Septate.
● Asexual spores: conidiophores; conidia (nonmotile).
● Sexual spores: Basidiospores, carried on the outer surface of a club-shaped cell called
the basidium.
● Common species: Smuts, rusts, puffballs, toadstools, mushrooms.

Deuteromycetes (Fungi Imperfecti)

● Mycelium: Septate.
● Asexual spores: conidiophores; conidia (nonmotile).
● Sexual spores: No sexual phase observed. Some members are classified as
ascomycetes or basidiomycetes.
● Common species: Aspergillus, Cryptococcus, Blastomyces, Histoplasma.
FUNGAL CULTURE
Gross colonial morphology is important in the identification of fungi.
Obverse: Yellow tan, rust colonies, colony surface is powdery or wooly-velvety
Reverse: cream, yellow, or light brown

TYPES OF MEDIA:
1. Potato Dextrose Agar (PDA)
- General purpose medium used for the isolation of fungi and molds. Sometimes
can be too rich and will encourage mold sporulation and pigment production
some dermatophytes

Components: Potatoes 200 grams

Agar 15-20 grams
Dextrose 15 grams
Distilled water 1000 mL

2. Corn Meal Agar (CMA)

- Used for growing a wide range of fungi, specially members of the
deuteromycetes (Fungi Imperfecti). Provides good balance of mycelial growth
and sporulation due to less easily digestible carbohydrate

Components: Cornmeal 20 grams

Agar 15-20 grams
Distilled water 1000 mL

3. Malt Extract Agar (MEA)

- Frequently used for culturing fungi inhabiting soil and wood. Malt extract lacks
peptone, and is useful for culturing Ascomycetes, where sporulation in some
species can be inhibited by the presence of peptone

Components: Malt Extract 20 grams

Agar 20 grams
Glucose 20 grams
Distilled water 1000 mL

4. Sabouraud Dextrose Agar (SDA)

- The standard medium for recovery and maintenance of a wide variety of fungi
commonly isolated in the clinical setting/laboratory

Components: Glucose 20 grams

Peptone 10 grams
Agar 20 grams
Distilled water 1000 mL

5. Czapek-Dox Agar (CZ)

- This medium is used for isolation and culture of saprophytic soil fungi

Components: Sucrose, Sodium nitrate, Magnesium sulphate, Potassium

Chloride, Iron sulphate, Dipotassium hydrogen phosphate, Agar, distilled water
Culture considerations:
1) Fungal cultures are incubated at room temperature
2) Visible growth requires from several days to several weeks
3) Cultures should be maintained in a high-humidity environment.

DIRECT OBSERVATIONS:
1. Saline wet mount - Viewing of fungal elements (e.g. hyphae, conidia, budding cells)
2. Lactophenol cotton blue wet mount - Stain and preserve fungal elements in culture
isolates
3. Potassium hydroxide (KOH) - Used to dissolve non-fungal materials in skin, nail, and
hair samples
4. Gram stain - Used to view yeasts
5. India ink - Stains capsules
6. Calcofluor white stain - Stains chitin

SLIDE PREPARATION:
1. Tease mount method
Dissecting needle is used to pull apart a fungal colony which is then placed on a slide. This
method may damage fungal structures such as conidia.

2. Cellophane tape method

Performed to transfer hyphae from the colony to a microscope slide for microscopic
examination.

3. Slide culture method

Uses a block of agar overlaid with a coverslip. Fungi are grown on the side of the agar block.
Following incubation, coverslip is removed and used for microscopic examination
CULTURE OF FUNGI

FUNGAL CULTURE IN SOLID PLATES

Principle:
Solid fungal growth on solid surfaces allows the examination and observation of gross
colonial morphology among different genera of molds.

Variations in colonial appearance are significant in fungal identification of filamentous

fungi. Also known as Agar block method.

Cultures:
Seven to 10 day old fungal cultures (grown using Sabouraud agar):
Aspergillus niger • Penicillium • Mucor • Rhizopus

Media:
Three Sabouraud agar plates
One potato dextrose agar plate

Equipment:
Bunsen burner, alcohol lamp, or microincinerator
Four test tubes containing 2 ml of sterile saline solution
Inoculating loop
Microscope

AGAR BLOCK METHOD

Label properly three Sabouraud agar plates as Aspergillus niger, Penicillium, and Mucor, and
label the fourth plate with PDA as Rhizopus.

Using an inoculating needle, aseptically cut out a small portion of the colony margin of the
provided fungal culture plate. Excised colony margin should be about 1 mm square. (Hyphal tip
transfers work best because this is the most active part of the culture)

Transfer the square of colony margin to a sterile fungal medium and place the agar block at the
center of the agar surface, withdraw the needle and flame-sterilized (Note: Necessary to kill all
adhering spores and hyphae)

Incubate inoculated plates at room temperature, 25ºC for 7-10 days. (Note: Do not invert the
plates).

After incubation period, study and examine the agar plates using a microscope (PART 2B:
Examination of molds)

(NOTE: Do not remove the Petri dish covers when viewing under the microscope)

A. Inoculating needle is flame sterilized

B. Small piece of colony is removed from the fungal culture
C. Transfer to a new dish containing culture medium
D. A plate containing a piece of the old culture at its centre
RIDDLE SLIDE CULTURE TECHNIQUE
Principle:
Riddell slide culture technique is used to allow examination of structural components of
molds with little disturbance as possible.

Spores are inoculated in the agar surface and incubated in a moist environment.
Direct microscopic observation can be performed without damaging the structure.

(Note: Fungal components are very fragile and even gentle use of the inoculating
loop/needle can result to the disruption of fungal components).

Cultures: Seven to 10 day old fungal cultures (grown using Sabouraud agar):
Aspergillus niger • Penicillium • Mucor • Rhizopus

Media: Sabouraud agar plates

Equipment: Bunsen burner, alcohol lamp, or microincinerator; Filter paper; U-shaped glass rod;
Microscope slides and coverslips; 95% ethanol

Slide Culture Preparation

Place a sheet of sterile filter paper in a petri dish using a sterile forceps (Note: Do this
aseptically)

Place the sterile U-shaped rod on the filter paper (Note: Rod can be sterilized by flaming)

Pour enough sterile water on the filter paper to moisten it.

Aseptically, using a forcep, place a sterile slide on the U-shaped rod.

Sterilize a scalpel by flaming, and cut an approximately 5 mm square block of Sabouraud agar
from the agar plate. Gently pick the agar block by inserting the scalpel and transfer to the center
of the slide.

Inoculate the four slides of the agar block with spores of mycelia of the fungus to be studied and
examined. (Note: Make sure to flame and cool the loop before picking up the fungal spores)

Place a sterile cover glass on the upper layer/surface of the inoculated agar block.

Cover the petri dish and incubate at room temperature for 48 hours. (Note: Remoisten filter
paper when necessary during incubation)

Following incubation, examine the slide under low power magnification for presence of hyphae
and spores. If growth is inadequate and spores are not present, extend incubation for another
24-48 hours

Examine each slide preparation under the microscope (PART 2B: Morphology of Fungi). Identify
structures such as mycelial mat, vegetative and reproductive hyphae, and spores. Record your
observations in the Lab report.

***After the fungi grow around the block, direct microscopic observation can be performed
without damaging the structure.
FUNGAL MORPHOLOGY

LACTOPHENOL COTTON BLUE WET MOUNTS

Principle:
Lactophenol cotton blue (LPCB) stains the fungal chitin in the cell wall and identification
of molds can be performed on the basis of their microscopic characteristics.

Fungal elements are stained intensely blue.

Lactic acid - preservative

phenol - disinfectant
cotton blue - stains chitin

We will use (1) Tease mount and (2) Scotch tape preparation to make the slide mounts.

1) Tease Mount Preparation

Aseptically, using a bent wire or inoculating needle, carefully pick a portion of the fungus
from the culture plate.

Transfer on a sterile slide containing a drop of lactophenol cotton blue.

Tease the hyphae of the fungi using the bent wire or inoculating needle.

Place a cover slip and examine under the microscope.

2) Scotch Tape Method

Take a clean microscope slice and place a drop of LPCB on it.

Touch the adhesive side of the scotch tapes on the surface of the colony at a point
between the center the periphery of the culture.

Fix the adhesive side on an area on the glass slide with LPCB.

Observe slide under the microscope

 EXAMINATION OF YEAST

Saccharomyces cerevisiae and Candida albicans

1. Aseptically make a wet mount slide of Saccharomyces cerevisiae using an inoculating

loop.

2. Stain the cells with methylene blue and observe under the microscope using high
power objective.

3. Look for vegetative cells and budding cells.

4. Draw what you have observed in the laboratory report template.

5. Observed prepared slides of Candida albicans under high power objective. You may
also use the OIO but do not force the Lens into position if it seems that it will hit the
coverslip.

6. Look for vegetative and budding cells.

COLONY EXAMINATION OF MOLDS

1. Obtain the culture plates of the molds from the agar block plates. Do not remove the
lid.

Examine the colony morphology, observe and record the color on both the top (Obverse)
and bottom (Reverse) surfaces.

colony texture:
Glabrous (leathery)
Velvety
Yeast-like
Cottony
Granular (powdery)

colony topography
Flat
Rugos (with radial grooves)
Folded
Crateriform
Verrucose (warty, rough)
Cerebriform (brain-like)

*** Examine the colony under a dissecting microscope and look for hyphae, rhizoids,
sporangiospores, and sporangia.
MICROSCOPIC EXAMINATION OF MOLDS

1. Observe and examine prepared slides of Penicillium and identify the following: hyphae,
conidiophores, metulae, phialides, and chains of conidia.

Observe and examine prepared slides of Aspergillus conidiophores and identify the following:
hyphae, conidiophores, phialides, and conidia.

Draw and label examples in the laboratory report sheet.

2. Examine prepared slides of Rhizopus and Mucor sporangia under the microscope.

Identify the following: Sporangiophores, sporangia, and sporangiospores.

Draw and label examples in the laboratory sheet.

EFFECTS OF CHEMICAL DISINFECTANTS ON BACTERIA

CONFUSING TERMS:
Chemical agents have been widely used to control microbial growth and they are widely used in
various fields.

Some are use directly on human tissues while other are applied on objects, but there are also
agents used as both.

There are terms for these agents depending on their usage and effectiveness but they get
oftenly interchanged.

DEFINITION OF TERMS:
Antiseptic
- chemicals applied to the skin or mucous membranes
- prevent microbial growth through inhibition or destruction

Disinfectant
- chemicals applied to the inanimate objects or surfaces
- prevent microbial growth through inhibition or destruction

Bactericide/Germicides kills bacteria/endospores.

Bacteriostatic agents inhibits bacterial growth but not necessarily kill them.

Viricide, sporicide, fungicides kill viruses, spores of fungi, respectively.

Sanitizers lower down microbial load to a safe level according to safety standards.

FACTORS AFFECTING EFFICIENCY

There are a number of factors that could affect the efficacy of a disinfectant
The type and number of microorganisms present
The material being disinfected
Concentration of disinfectant
Time allowed for the disinfectant to act (contact time)
Temperature of the disinfectant

The disinfectant can affect the integrity of some parts such as the cell wall, cell membrane or
cytosol, structural proteins, etc. which contributes to their mode of action.

EXPERIMENT:
200 ul b. Subtilis → vortex → time 1 minute → disinfectant + b.s → 100 ul of mixture to Tryptic
soy → vortex → incubate 37 deg for 48 hrs → observe for turbidity

CONTROLS
Disinfectant → 100 ul → vortex → incubate 37 deg for 48 hrs → observe for turbidity

Pure culture of bacteria → 100 ul → vortex → incubate 37 deg for 48 hrs → observe for turbidity