LABORATORY | MICROSCOPY & EXERCISE 1: FUNGAL STRUCTURE

OUTLINE • HPO: examination of microorganisms suspended in fluid

I. MICROSCOPY • OIO: examination of stained smears
a. Parts & Uses of a Microscope
II. EXERCISE 1: FUNGAL STRUCTURE
Reference: Mycology & Virology Laboratory Manual
Prof. Viole N. Bascao, RMT, MSMT, MAEd
MICROSCOPY
• MICROSCOPE: A laboratory instrument used to enlarge images not
typically seen by the naked eye
PARTS AND USES OF A MICROSCOPE
PART FUNCTION
EYE PIECE/OCULAR Used to observe or view the specimen
DIOPTER Figure 1.1: Mold
Used to compensate the view of the user
ADJUSTMENT RIM
Holds and connect the eyepiece and
HEAD
objective lenses of the microscope
Support of the microscope; connects the
ARM
body tube to the base of the microscope
STAGE It is where the slide is placed
REVOLVING
It holds the different objectives
NOSEPIECE
Scanner (4x), Low power (10x), High Figure 1.2: Yeast
OBJECTIVE LENSES
power (40x), Oil immersion (100x) EXERCISE 1: FUNGAL STRUCTURES
STAGE CLIP Used to hold the slide in place • Fungi: a filamentous, non-photosynthetic, eukaryotic
Left to right movement (X-axis) microorganism with a heterotrophic nutrition.
X & Y AXIS KNOB
Top to bottom movement (Y-axis) o Hypha = basic cellular unit; extends by the tip growth and
COARSE Used for the initial focusing of the multiplies by branching creating a mycelium.
ADJUSTMENT KNOB specimen; used in scanner and LPO ▪ Contains: nuclei, mitochondria, ribosomes, golgi and
FINE ADJUSTMENT Used to provide a finer image of the membrane-bound vesicles w/ a plasma-membrane
KNOB specimen; used in HPO and OIO bound cytoplasm
CONDENSER Concentrates light o Septae = cross walls that separate older hyphae parts to
IRIS DIAPHRAGM Regulates/adjusts light intensity younger hyphae parts
ILLUMINATOR Source of light • Yeast: multiplies via binary fission/budding.
REOSTAT Used to adjust light intensity MATERIALS:
BASE Supports the entire microscope • Slides • Microscope
Table 1.1: Parts of a Microscope o Mold (Aspergillus spp., Sporangia, • Immersion oil
RESOLUTION/ Ability to differentiate two lines/points in an Rhizopus) • Lens paper
RESOLVING object; higher the resolution, the finer the o Yeast (Saccharomyces)
POWER image PROCEDURE:
WORKING Distance between the objective lens and the 1. Examine the prepared slides using:
DISTANCE slide o LPO & HPO for Molds
Light-gathering ability and resolution; higher o HPO & OIO for Yeast
NUMERICAL
the numerical aperture, better light- 2. Draw & label the different fungal structures
APERTURE
gathering capability, closer the lens
Ability of microscope to remain in focus
PARFOCAL
even after changing objectives
TOTAL
Eyepiece (10x) x eyepiece magnification
MAGNIFICATION
NUMERICAL
OBJECTIVE MAG. TOTAL MAG.
APERTURE
LPO 0.25 10x 100x
HPO 0.65 40x 400x
OIO 1.25 100x 1000x
Table 1.2: Objective Lenses Figure 2.1: Aspergillus spp.

USES OF THE DIFFERENT OBJECTIVES IN

MICROBIOLOGY
• LPO: examination of large organisms

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 MICR_112LAB: Microscopy & Exercise 1

Figure 2.2: Sporangia

→ Sporangium (black circle) = “sac-like” structure that holds the
spore (Sporangiospore)
→ Sporangiophore = a type of hyphae

Figure 2.3: Rhizopus

→ Rhizopus produces a zygote; it is the sexual reproduction of
sporangium
→ (red circle) presence of hyphae on both sides w/c would lead
to production of a zygote

Figure 2.4: Saccharomyces

→ Distinguishing characteristic of yeast = bud formation

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 LABORATORY | EXERCISE 2: FUNGAL CULTURE & ISOLATION
OUTLINE MATERIALS & PROCEDURES
I. FUNGAL CULTURE & ISOLATION MATERIALS:
a. Specimens in Mycology
b. Materials & Procedures • 2 Sabouraund Dextroe Agar • Forceps
c. Data & Results o General culture medium for • Hair strand
Reference: Laboratory Manual, Lecture discussion fungi (specimen)
FUNGAL CULTURE & ISOLATION • Bacticinerator
SPECIMENS IN MYCOLOGY PROCEDURE (from ppt, lab manual)
• Blood • Subcutaneous (tissue 1. Collect the specimen (cutaneous specimen)
• Bone marrow aspirate biopsies & lesions/exudate) 2. Sterilize the forceps
o Iliac crest, between L3 & • Respiratory specimens 3. Place the hair strand on the center of each SDA plates / Potato
L4 o sputum, bronchial dextrose Agar (PDA)
o Px in fetal position washings & 4. Gently press the specimen using sterilized forceps/inoculating
• CSF transtracheal aspirate needle onto the SDA for better observation of the colonial
• Cutaneous • Throat swab growth
o Skin scrapings, Hair & • Vaginal & Cervical 5. Label the SDA with the ff:
Nails (has high keratin • Prostatic secretions o Type of specimen,
content) o Date and Time of inoculation,
• Mucocutaneous o Incubation temperature,
(tongue/cheeks) o Initials of RMT
• Urine 6. Incubate one SDA at room tempt & the other at 37°C for 3-5
days or until visible colonial growth appears
7. Examine the culture everyday & look for presence of fungal
colonies (yeast/mycelial form)
8. Record all the observations
PROCEDURE – SKIN SCRAPINGS
1. Clean the area with a 70% alcohol (pad).
o Minimizes contamination & aid during microscopy if
greasy ointments/powders have been applied
2. Using a sterile scalpel blade, scrape the edge of the lesion &
collect the skin scrapes into a dermapak.
o Collect as much specimen as possible to allow a full
investigation to assist in the correct diagnosis.
o If there is no available dermapak, a clean piece of paper
Figure 1.1: Specimens for may be used.
Mycology 3. Fold up, seal & label properly with the px’s complete name,
date & time of collection.
PROCEDURE – NAIL CLIPPINGS
1. Clean the area with 70% alcohol.
2. Using a sterile nail clippie, cut the infected nails (discolored,
thickened, dystrophic, brittle parts of the nail)
o if possible, collect the subungual debris in addition to nail
clippings
3. Place the collected sample using a dermapak/clean paper
Figure 1.2: Mycolytic culture
vials 4. Fold, up, seal & label.
PROCEDURE – HAIR STRAND
1. Pluck hairs from the infected area using sterile forceps.
Infected hairs come out easily
2. The sample should include hair roots, contents of plugged
follicles & skin scales. If the hair is too long cut with sterile
• For skin scraping use sterilized scalpel, not the blade part, or scissors. A hair sample cut with scissors is unsatisfactory as the
use a sterilized glass slide from inner to outer focus of infection is below/near the surface of the scalp
• For the hair, woods lamp can be used. Px is situated in a dark 3. Fold, seal, & label.
room, the woods lamp is placed 12 inches above the infected
area. Fluorescense is observed, which indicates infection.

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 MICR_112LAB: Exercise 2: Fungal Culture & Isolation

DATA & RESULTS

Figure 1.3: Colony at 37°

Figure 1.4: Colony at room temperature

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 LABORATORY | EXERCISE 3A: CULTURINNG TECHNIQUES
OUTLINE
I. CULTURING TECHNIQUE
a. Material & Procedures
b. Data & Results
Reference: Mycology & Virology Manual; Laboratory discussion Figure 1.2: Wet Mount
Technique
DIRECT EXAMINATION OF FUNGI:
CULTURING TECHNIQUES
• Adhesive tape preparation = used by most laboratories; can easy,
quick & sufficient to make an identification for most fungi
• Wet mount = offered by some laboratories TEASE MOUNT PREPARTION
• Microslide culture method = used for greater detail of the 1. Sterilize two inoculating needles
morphologic feature required for identification 2. Select and get a portion of a fungal colony between the center
MATERIALS & PROCEDURES and the peripheral part
• Fungal colony • Cover slip 3. Place the colony on a clean glass slide and using the two
• Inoculating needle • Scotch tape inoculating needles (dissecting needle) gently spread the colony
• Bacti-cinerator • Glass slide by teasing/tearing it apart
• LPCB (Lactophenol Cotton Blue) o Disadvantage: possible destruction of the hyphae due to the
pressure of separating thru teasing/tearing
4. Place a drop of LPCB then place a coverslip
5. Examine microscopically using LPO and HPO

Figure 1.3: Tease Mount

Preparation

Figure 1.1: Exposed SDA in RT for 1hr

• After 1hr incubate the agar and wait a couple of days for SCOTCH TAPE/CELLOPHANE TAPE
growth. PREPARATION
• 1-3 days = rapid grower 1. Cut the ordinary tape with the same size of the glass slide
• 5-9 days = intermediate/moderate grower 2. Touch the adhesive portion of the tape on the top of the colony
• 1 week – 8 months = slow grower and let the aerial hyphae adhere to the tape
• Q: How do you describe colonial growth of molds? o Disadvantages: recovery of aerial hyphae only; risk of bubble
Fuzzy/Wooly, Cottony, Velvety formation
3. Gently remove the tape and place onto a drop of LPCB on the
WET MOUNT TECHNIQUE glass slide
1. Using an inoculating needle bent at a 90-degree angle, cut out a 4. Examine microscopically using LPO and HPO.
small portion of an isolated fungal colony
2. Place the agar block on a clean glass slide then add a drop of
lactophenol cotton blue (LPCB)
3. Put a cover slip over the agar block and gently apply pressure
Figure 1.4: Scotch tape
using the handle of the inoculating needle to disperse the colony
preparation
and the agar
4. Examine microscopically using LPO and HPO
o For fresh microorganisms, highest magnification is HPO

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 MICR_112LAB: Exercise 3A Culturing Techniques

DATA & RESULTS

Figure 1.5: Wet mount (left) LPO (right) HPO

Figure 1.6: Scotch tape (left) LPO (right) HPO

Figure 1.7: Tease mount (left) LPO (right) HPO

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 LABORATORY | EX. 3B: DIFFERENT STAINS & EX. 4: SLIDE CULTURE METHOD
OUTLINE • Bacticinerator
I. DIFFERENT STAINS PROCEDURE
a. 10-20% KOH (1st method)
b. India Ink
c. Lactophenol Cotton Blue
1. Sterilize the inoculating loop.
II. SLIDE CULTURE METHOD 2. Place 1 drop of India Ink on one end of a clean glass slide.
Laboratory discussion, Mycology & Virology Manual 3. Get a loopful of colony and mix with the stain.
Prof. Viole N. Bascao, RMT, MSMT, MAEd 4. Spread the mixture and make a smear.
5. Label the slide.
DIFFERENT STAINS 6. Allow the smear to dry (air dry).
• Provide the first microbiologic proof of the etiology of disease in 7. Examine under HPO and OIO.
patients w/ fungal infection 8. Record the results.
10-20% KOH PREPARATION (2nd method)
• Principle: Potassium hydroxide preparation is used in the initial 1. place one drop of stain + one drop of) of organism on the glass
examination of keratized tissue suspected of fungal infection. slide
• KOH dissolves keratin to facilitate observation 2. organism should be coming from a broth medium
MATERIALS 3. Then cover with cover slip
• Hair strand (specimen) • Forceps 4. Observe up to HPO only
• 10-20% KOH • Glass slide
• Alcohol lamp • Cover slip
PROCEDURE
1. Sterilize the forceps.
2. Place the specimen (hair strand) on the glass slide. Figure 1.3 India
3. Add a drop of 10-20% KOH. Ink – HPO
4. Put cover slip. Label the preparation.
5. Gently warm the glass slide by passing over the flame 3-5
times.
o To dissolve keratinized cells
6. Allow the specimen to clear for 10-20 mins.
7. Examine under LPO and HPO.
8. Record the results.

Figure 1.4 India

Ink - OIO

Figure 1.1 10-20%

KOH – LPO

LACTOPHENOL COTOTN BLUE PREPARATION

• Used commonly in the laboratory
• Colorless nail polish – used to preserve the slide for future
observations
MATERIALS
• Fungal Colony (specimen) • Inoculating needle
• LPCB • Glass slide
Figure 1.2 10-20% • Bacticinerator • Cover slip
KOH – HPO
PROCEDURE
1. Sterilize the inoculating needle.
2. Cut a portion of the colony and place on a clean glass slide.
3. Add 1 drop of LPCB and put cover slip.
4. Gently press the cover slip to spread the agar and stain.
INDIA INK PREPARATION 5. Label the glass slide.
• Nigrosine dye 6. Examine under LPO and HPO.
• Stains the background, specimen/organism unstained 7. Record the results.
MATERIALS
• Candida albicans (specimen) • Inoculating loop
• India ink • Glass slide

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 MICR_112LAB: Exercise 3B Different Stains & Exercise 4 Slide Culture Method

Figure 1.5 LPCB Figure 2.1 Slide

– LPO Culture - LPO

Figure 1.6 LPCB

Figure 2.2 Slide
– HPO
Culture - HPO

SLIDE CULTURE PREPARATION

• Microculture preparation/Subculturing method
• For observation of aerial & vegetative hyphae
• Direct inoculation of fungi on a slide
• Fungus will grow on the glass slide
• Most accurate method to preserve and observe fungal
microstructure
MATERIALS
• Specimen: Fungal Colony • Filter paper/gauze pad
• SDA (for agar block) • LPCB
• Bacticinerator • Glass slide
• Forceps • Coverslip
• Inoculating needle • Applicator stick
• Petri dish (glass) • Sterile Distilled Water
o prevent loss of moisture
PROCEDURE
1. Place sterile gauze pad into sterile petri dish.
2. Place applicator sticks and carefully pour sterile distilled water
into the gauze pad.
o distilled water will help in maintaining moisture
3. Sterilize a glass slide and place it on top of the applicator stick
4. Cut the agar block approx. 1.5 x 1.5cm and place the agar block
on the center of the glass
o Another option: using a sterilized test tube, cut the ager by
placing the opening of the test tube forming a round agar
5. Cover the petri dish
6. Inoculate the fungus into the four corners of the agar block
using right angle wire
o Stab the corners
7. Flame a coverslip using forceps (2-3 seconds). Let it cool for a
few seconds and place on top of the afar block
8. Incubate at RT (25-27C) for approx. 3-4 days or until visible
growth
o Label is placed on the cover of the petri dish
9. After incubation, remove coverslip and heat-fix the fungus by
passing the coverslip over the flame 2-4 times
10. Place the coverslip on a new slide with 1 drop of LPCB
11. Examine microscopically
o Observe presence of microconidia, macroconidia and
other fungal structures

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 GERM TUBE TEST
MICR_112: MYCOLOGY AND VIROLOGY
LABORATORY
PROF. VICTOR PERLAS
OUTLINE PROCEDURE
I. Introduction 1. Pipet 0.5 ml of serum into a test tube.
II. Materials 2. Suspend small inoculums of yeast cells obtained
III. Procedure from an isolated colony. (A large colony may
IV. Data & Results
produce a false-negative result)
3. Incubate the tubes at 37°C for 2 to 3 hours.
INTRODUCTION
4. Place a drop of the suspension on a glass slide
● Candida spp. is the most common cause of
and apply a coverslip.
opportunistic mycoses worldwide.
5. Examine microscopically (LPO and HPO).
● It is also a frequent colonizer of human skin and
6. Record and draw the results.
mucous membranes.
● It is a member of the normal flora of skin, mouth,
DATA & RESULTS (Candida albicans)
vagina, and stool. As well as being a pathogen
and a colonizer, it is found in the environment,
particularly on leaves, flowers, water, and soil. LPO HPO
● The genus Candida includes around 154 species.
Among these, six are most frequently isolated in
human infections.
● Candida albicans is the most abundant and
significant species, Candida tropicalis, Candida
glabrata, Candida parasilosis, Candida krusei, and
Candida lusitaniae are also isolated as causative
agents of Candida infections.
● Candida albicans can be presumptively identified
based on the production of germ tubes when
placed in a liquid nutrient such as rabbit serum.

GERM TUBE TEST

Principle: C. albicans produce germ tubes,
pseudohyphae & blastoconidia from their yeast cells
when placed in a liquid environment (serum) and
incubated at 35-37ºC for 3 hrs.
Results:
– Positive (+) = C. albicans & C. dubliniensis
– Negative (-) = C. tropicalis & C. glabrata

QUALITY CONTROL
– Positive Control (+) = Candida albicans (ATCC
10231) – Negative Control (-) = C. tropicalis (ATCC
13803)
= C. glabrata (ATCC 2001)

Materials
● Unknown organism
● Rabbit Serum
● Inoculating loop
● Test tube
● Glass slide

1 MENDOZA, SKY
 IDENTIFICATION OF BREAD
MOLD/BLACK MOLD
MICR_112: MYCOLOGY AND VIROLOGY
LABORATORY
PROF. VICTOR PERLAS
OUTLINE
I. Introduction
II. Materials DATA & RESULTS
III. Procedure
IV. Data & Results LPO HPO

INTRODUCTION
● Have you ever seen a bread mold - a black, fuzzy
substance growing on bread?! If so, you have
seen a sporangium fungus.
● Sporangium fungi are fungi that produce spores
in sporangia. Sporangia are structures, found on
the tips of hyphae that make spores. You can see
that the phylum gets its name from these
structures.
● Bread mold produces spores within sporangia
that stick up above the bread. Each spore can
form a new bread mold. Hyphae grow into bread.
● The mold takes in food and water by means of its
hyphae.
● Molds grow best in warm, moist, and dark places,
but they can grow also in cold places, such as
refrigerators.
● Growth of molds on food can be prevented by
cleanliness, drying, and the use of chemicals,
● Chemicals are added to bread to delay the growth
of molds. Fruits such as raisins and apricots are
preserved by drying.
Materials
● Bread
● Plastic bag/Ziploc
● Water
● Glass slide

PROCEDURE
1. Moisten a piece of bread with tap water and
place it in a sealed plastic bag.
2. Incubate the preparation at room temperature
for about one week or until molds appear
3. Using the adhesive tape method, obtain
samples and place them on a slide with
lactophenol cotton blue (LPCB).
4. Examine microscopically using low power
objective and high power objective.
5. Record and draw the results.

1 MENDOZA, SKY
 HAIR PERFORATION TEST
MICR_112: MYCOLOGY AND VIROLOGY
LABORATORY
PROF. VICTOR PERLAS
OUTLINE 6. If there is fungal growth, make slide mounts
I. Introduction using LPCB or KOH.
II. Materials 7. Examine microscopically and record the results.
III. Procedure

INTRODUCTION
● Trichophyton is a dermatophyte that inhabits the
soil, humans, or animals. Related to its natural
habitats, the genus includes anthropophilic,
zoophilic, and geophilic species.
● It is one of the leading causes of hair, skin, and
nail infections in humans.
● Most of the Trichophyton species have
teleomorphic forms and these teleomorphs are
classified in the genus Arthroderma.
● Trichophyton is a keratinophilic filamentous
fungus. It possesses several enzymes, such as
acid proteinases, elastase, keratinases, and other
proteinases that are the major virulence factors of
these fungi.
● The genus Trichophyton has several species.
Most common are T. mentagrophytes, T. rubrum,
T. schoenleinii, T .tonsurans, T. verrucosum, and
T. violaceum.
● The most common species that can be isolated in
the laboratory are T. mentagrophytes and T.
rubrum. In order to differentiate the two, some
tests should be performed such as the hair
perforation test, urease test, and their reactions
to Trichophyton agars.

Materials
● Fugal colony
● Petri dish
● Distilled water
● Alcohol lamp
● LPCB or KOH

PROCEDURE
1. Place several short strands of sterilized human
hair in a sterile petri dish.
2. Add 25 ml of sterile distilled water and 2-3 drops
of 10% yeast extract.
3. Inoculate plates with several small fragments of
the test organism.
4. Label the petri dish and incubate at room
temperature for 3 to 5 days. (maximum of 1
week )
5. Examine the plates every day and note for
fungal growth.

1 MENDOZA, SKY
 HAIR BAITING
MICR_112: MYCOLOGY AND VIROLOGY
LABORATORY
PROF. VICTOR PERLAS
OUTLINE depressions on the soil until you can see the
I. Introduction bottom of the dish
II. Materials 4. Moisten the soil by adding small amount of
III. Procedure sterile water.
IV. Data & Results 5. Using sterilized forceps, sprinkle the hair onto
the soil allowing some to fall into the
INTRODUCTION depressions.
● The Cutaneous Mycoses infect the dead, horny 6. Cover the plate and incubate at room
layer of the skin, hair, and nails to a greater temperature for 5 days to 1 week.
degree than the superficial mycoses. 7. After incubation, you will notice a white, fuzzy
● These fungi secrete an enzyme called keratinase, mycelium will start to grow on the pieces of hair.
which digests keratin. Since keratin is the primary 8. To observe the fungi in detail, get a piece of hair
structural protein of the skin, nails, and hair, the and place it on a glass slide with a drop of
digestion of keratin manifests as scaling of the lactophenol cotton blue (LPCB).
skin, loss of hair, and crumbling of the nails. 9. Examine it microscopically using low power
● The cutaneous mycoses is also known as objective and high power objective. Look for the
dermatophytosis, a clinical entry caused by any presence of hyphae or spores which will help in
31 recognized species of pathogenic and the identification of the fungus.
taxonomically related fungi (dermatophytes) of the 10. Draw your observations on the space provided.
genera are the following:
○ Trichophyton (infect the skin, hair and nails) DATA AND RESULTS
○ Microsporum (infect the skin and hair)
○ Epidermophyton (infect the skin and nails).
● Dermatophytes are groups of fungi that infect the Microsporum gypseum
outermost surfaces. Because they are
"keratinophilic", it is possible to allow fungi to grow
on sterile hair since keratin is a major component
of hair
● Hair Baiting is one of the tests which can be used
to differentiate M. gypseum to M. audounii and M.
canis.

Materials LPO HPO

● Soil
● Hair Strand
● Forceps
● Petri dish
● Bactecinerator
● Microscope
● LPCB

PROCEDURE
1. Obtain some strands of human hair. Place it in a
sterile flask with acetone and shake for 20 to 30
seconds. (this will remove the fats and dirt from
the hair)
2. Discard the acetone and let the hair dry. Cut hair
into pieces about 1 to 2 cm long
3. Place some soil in a Petri dish, enough to cover
the bottom part, with your fingers, make 4 to 5

1 MENDOZA, SKY