Specimen Collection and Transport MYCO&VIRO

Prof. Esmeralda Abling LE 02

31 January 2022 TRANS 01

OUTLINE
I. Specimen Collection
II. Samples commonly submitted to mycology laboratory
A. Hair
B. Skin
C. Nails
D. Blood
E. Bone Marrow
F. Cerebrospinal Fluid
G. Abscess Fluid, Wound Exudates, and Tissue
H. Respiratory Tract Secretions
I. Corneal Scrapings
J. Urine
K. Vagina
III. Laboratory Diagnosis of Mycoses
A. Direct Microscopic Examination
B. Special Tests
I. SPECIMEN COLLECTION
• Sterile equipment and aseptic techniques to avoid the
introduction of microorganisms during invasive procedures
• Adequate amount of specimen to decrease the chance of false
negative results
• Sample from the area most likely affected by the organism
• Proper specimen labelling.
NEED-TO-KNOW
• Specimen collection is critical since errors committed cannot
be corrected and will require collection of a new specimen
• The primary criterion for accurate diagnosis of mycotic
infection is the proper collection of appropriate samples for
testing
• Volume of specimen required for fungal cultures usually
exceed and is greater than those used in bacteriology
® Several types of specimens need to be pre-treated or
concentrated before inoculation(plating) in order to
maximize the recovery of the fungi
• Best specimen to be submitted must come from the active
site of infection
® The choice of the correct specimen in Mycology will
depend on the clinical presentation of the patient and
the organ systems affected
• Improper specimen labelling or patient identification is the
most common source of pre-analytical error
• Specimen container must contain the pertinent information
of the patient
® Patient’s name
® Hospital number
® DOB
® Time and Date of collection
® Type of specimen
® Test requested
• Usually, the specimen container is labelled with 2 patient
identifiers (most common)
® Patient’s name and Hospital number / Patient’s name
and DOB
• Transport specimens in leak proof sterile container and
processed as soon as possible.
• Mold cultures and clinical specimens must be handled
in class II BSC because we want to provide protection to the
specimen, environment, and laboratory worker from the
dissemination of the conidia which are airborne
NEED-TO-KNOW
• Many pathogenic fungi grow slowly, and so any delay in the
processing decreases the probability of isolating the
causative agent, as a result of the overgrowth by
contaminants
• Cultures of organism suspected of being pathogens should
be sealed with tape to prevent lab contamination
® Should be autoclaved as soon as definitive
identification is made
Universal Precaution
• “Treat blood and other body fluids as if they contain blood-
borne pathogen”
Conidia
• Asexual reproductive structure
• Screw capped tubes are preferred to Petri dishes for
recovery of fungi. Petri dishes are not recommended
® Release of airborne conidia is less likely
® Reduce dehydration of media
® Easy handling and storage; takes up less space for
incubation
NEED-TO-KNOW
• Petri dish is not recommended because the agar in the petri
dish dehydrates/dries easily due to the extended incubation
time that is required in fungal cultures. But it still has
advantages:
® Larger surface area for colony isolation
® Easier to manipulate when making preparations for
microscopic examination
II. SAMPLES COMMONLY SUBMITTED TO MYCOLOGY
LABORATORY FOR CULTURE
A. HAIR
• Samples from the scalp include skin scales and plucked hairs.
• Hairs infected with fungi (e.g. Microsporum audouinii – causes
Tinea Capitis infection) fluoresce/will give off light using Wood
lamp
NEED-TO-KNOW
• Cut hairs are not suitable for direct examination
• We have to use sterile forceps in pulling/plucking the hair:
® At least 10-12 hairs with the roots attached or with the
base shaft intact
• Wood Lamp – source of ultraviolet light and is used to
detect fluorescence in the skin and hair.
B. SKIN
• Samples from the outer edge of skin lesions are obtained by
scraping with scalpel blade or curette
• Choose the most recent for scrapings in case of multiple
lesions
NEED-TO-KNOW
• Skin is scraped from the outer edge of the skin lesion
® Because it contains the greatest amount of viable
fungus
• In cases of multiple lesions, you have to choose the most
recent for scrapings
C. NAILS
• Scraping of outermost layer of infected nail with scalpel or
curette
• Deeper scrapings, debris from under the edges of the infected
nails, and nail clippings are also suitable for culture.

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 2.01 Specimen Collection and Transport
NEED-TO-KNOW
• May be submitted as scrapings, cuttings, and occasionally,
as a complete nail.
• Discolored or brittle parts of the nail must also be included
in the collection
HAIR, NAILS, AND SKIN
• For dermatophyte culture
• Sites are initially cleaned with 70% alcohol to remove surface
contaminants
• Curette can also be used to collect specimens from young
children and from awkward sites such as interdigital spaces
• Samples should be placed in sterile petri dish and transported
to the lab. Storage at 4c is not recommended since some
dermatophytes are susceptible to cold temperature.
• After samples are received in the lab;
® KOH wet mount is prepared
® Samples are inoculated in Saboraud Dextrose Agar (SDA)
containing antibiotics:
o Chloramphenicol
o Gentamicin
o Cycloheximide
• Mycosel agar can also be used for inoculation (contains
chloramphenicol and
cycloheximide)
NEED-TO-KNOW
• Transport should be at room temperature
® Fungi are resilient/flexible but some of them are
affected by temperature above 37 degrees and below
10 degrees Celsius
• Hair, nails, and skin are particularly sensitive to cold
temperature
• SDA – standard culture medium used for the recovery of
fungi
• Cultures should be incubated for a minimum of 21 days at
30 degrees Celsius before reporting the results as negative
D. BLOOD
• 10 ml blood should be collected in SPS tube before they are
placed in blood culture bottles or can be inoculated directly in
blood culture bottles
® BACTEC, an automated blood culture system, is used for
the recovery of yeasts, except Malassezia spp.
• Recovery of fungi from blood samples may be enhanced using
Biphasic culture bottle
® Contains slant of Brain Heart Infusion Agar (BHIA) and
60-100ml of BHI broth
® 1:10 to 1:20 (blood broth ratio)
® Minimum of 5ml of blood is required for each culture bottle
® Culture bottles should be incubated at 30 degrees Celsius
and maintain for 4 weeks
• SDA with chloramphenicol and gentamicin may be used for
primary isolation of fungi in blood samples (Inoculation)
• Giemsa, Gram and Periodic Acid Schiff (PAS) are used for
stained smears, for direct microscopic examinations.
• Aside from culture bottles, we can use Isolator tubes. Isolator
tubes enhance fungal recovery (Histoplasma capsulatum and
other filamentous fungi) from blood and bone marrow
specimens
® Contain agents which lyse WBC and RBC in the blood
which then releases the microorganisms
® Centrifugation of the tube concentrates the organism
before culturing
o The concentrate is inoculated in appropriate culture
media and incubated at 30 degrees Celsius for 21
days
MV
NEED-TO-KNOW
• The use of blood culture provides an accurate method for
determining the cause of disseminated fungal infection
® Blood from septicemic patients can harbor known
pathogenic and opportunistic fungi, the most common
being is Candida
Figure 1. Automated blood culture system called BACTEC (Left). Biphasic culture
bottle (Middle). Isolator tubes (Right).
E. BONE MARROW
• The specimen from the bone marrow is obtained by means of
aspiration
• Bone marrow aspirate may be collected in a heparinized
syringe or isolator tube
® This bone marrow aspirate is then subjected to Giemsa
staining
® Inoculate in Saboraud Dextrose Agar (SDA) with
chloramphenicol and gentamicin and Brain Heart Infusion
Agar (BHIA) supplemented with 5% sheep blood.
® An alternative to this is to place the bone marrow aspirate
in an isolator tube and to process it as a blood culture
NEED-TO-KNOW
• These bone marrow samples are used to determine the
cause of disseminated fungal infections
® Such as fungi that causes septicemia in patients. The
most common being Candida
F. CEREBROSPINAL FLUID
• Collected by means of lumbar tap or lumbar puncture and
should be processed immediately
• Collected in 3 sterile tubes
® The second tube goes to the microbiology section
• 3-5 ml is adequate for the examination
• If there is a delay in the processing, samples should be kept at
room temperature or incubated at 30°C.
• Sample should be concentrated by centrifugation before
inoculation in a solid culture media
® The supernatant is reserved for cryptococcal antigen
testing
• Aside from inoculation in a solid culture media, you can also
perform:
® Cryptococcal antigen testing
® India Ink preparation
® Inoculate in SDA and BHIA supplemented with 5% sheep
blood.
NEED-TO-KNOW
The sediment is processed as follows:
• For direct microscopy, use a drop of the sediment to make
an India ink preparation. Resuspend the remaining
sediment in 1-2ml of CSF and inoculate in media with no
antibacterial or antifungal agents such as SDA and BHIA
supplemented with blood. Incubate at 35C and maintain
cultures for at least 4 weeks
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 2.01 Specimen Collection and Transport
G. ABSCESS FLUID, WOUND EXUDATES, AND TISSUE
• Abscess fluid and exudate from wounds are examined for the
presence of granules using dissecting microscope.
• If no granules are present, the material may be plated directly
onto the media.
• Tissue should be gently minced before culture/ inoculation
® SDA with chloramphenicol and gentamicin
® BHIA supplemented with 5% sheep blood
® KOH preparation
® Calcofluor white preparation
® Staining (Hematoxylin & Eosin, Methenamine Silver stain,
Periodic Acid Schiff)
• Aside from mincing, grinding of tissue has also been
recommended
® However, this process might destroy some fungal
elements
NEED-TO-KNOW
• In cases of large sections of tissue are submitted to the
laboratory, suspicious areas such as purulent or discolored
sections are selected for mincing and grinding before being
placed in a solid culture
• It is essential that the histopathology department also
receives a portion of the tissue in formalin for rapid frozen
section with staining by H&E. Methenamine Silver Stain and
Periodic Acid Schiff
H. RESPIRATORY TRACT SECRETIONS
• The most common specimens submitted are sputum and
transtracheal/tracheal aspirate
® Which are lower respiratory tract secretions
• Collect early morning sputum
® The patients should obtain the specimen from a deep
cough shortly after waking up in the morning and before
eating in order to reduce the risk of contaminating the
sample with food
® Ask the patient first to gargle to avoid contaminating the
sample with food particles
® Should be collected in a sterile container
• Viscous specimens such as tracheal aspirate can be digested
with a use mucolytic agent, which is N-acetyl-L-cysteine and
then concentrated before it is inoculated in solid culture media
• Specimens may be stored at room temperature if processing is
completed within 2 hours
• If processing is delayed, specimens should be refrigerated at
4°C.
• The sample is used to make:
® KOH preparation
® Gram stain
® Inoculate in SDA with chloramphenicol and gentamicin
and BHIA supplemented with blood.
o Any bits of blood, pus or necrotic material should be
plated directly onto the media
• Incubate culture media at 35C and maintain cultures for 4
weeks
I. CORNEAL SCRAPINGS
• For patients presenting with atypical lesions or ulcers, obtain
corneal scraping using surgical blade or calcium alginate swab
• Collected using surgical blade or calcium alginate swab
® Inoculate directly on SDA at the time of collection and
apply the remaining material to glass slides for staining
® Examined under the microscope using smears that are
stained with:
o Gram stain
o Lactophenol cotton blue (LPCB)
• The swab that is pre-moistened is rolled over the onto the
conjunctiva
MV
® Even if only one eye is infected, if possible, obtain two
separate swabs from each eye
® So that the other eye that is not infected will show the
indigenous microflora and serve as the control
• Withdraw the use of antibiotics before taking these corneal
scrapings or for any other clinical specimens
® You have to take the sample before the initiation of
antimicrobial therapy
® Other wise you will obtain false negative results
• Store at room temperature if there is any delay in the transport
Figure 2. Surgical blade (left). Calcium alginate swab (right)
J. URINE
• First morning urine obtained by midstream clean catch
method or catheter
• Refrigerate specimens at 4C for no longer than 12-15 hours if
there is delay in processing (cannot be competed within 2
hours)
® Urine is a very good medium for the replication of the
bacteria and fungi
® If you leave the specimen at room temperature, this will
allow the growth of contaminant
® It is recommended to process the specimen immediately
• Centrifuge urine and use the sediment to perform
® KOH preparation
® Inoculate in SDA with gentamicin and chloramphenicol
o Used because the urine is often contaminated with
gram negative bacteria
o Used to ensure the recovery of the fungi
• Centrifuge the urine for 10-15 minutes at 200rpm.
® KOH preparation
® Inoculate in SDA with gentamicin and chloramphenicol
and incubate at 35C.
K. VAGINA
• Vaginal swabs should be transported to the laboratory within 24
hours of collection using transport swabs which must be kept
moist in sterile tubes.
® This method of collection provides specimen suitable for a
wet preparation
• Selective and inhibitory agars should be plated.
• Vaginal cultures should be screened for yeasts and incubated
at 30°C for 7 days.
• Fungal cultures will require a long period of incubation, usually
3-4 weeks
® What happens is that the agar, should you use the petri
dish, will be dehydrated and will allow the growth of
contaminants due to the extended incubation time
® This solid culture media contains antibiotics to inhibit the
growth of bacteria and other fungi
NEED-TO-KNOW
• Recommended temperature for the transport of these
samples is at room temperature
® Because some of these fungi are sensitive to cold
temperature
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 2.01 Specimen Collection and Transport
Figure 3.
III. LABORATORY DIAGNOSIS OF MYCOSES
A. DIRECT MICROSCOPIC EXAMINATION
The two purposes of direct microscopic examination:
Presumptive diagnosis
• Provides a rapid or an initial report to the doctor which may
result to early initiation of treatment
Provides evidence of infection despite negative culture
results
• This may happen in specimens from patients receiving anti-
fungal therapy. These anti-fungal agents may inhibit the growth
of microorganisms in vitro, even though the infection can still be
present in the patient
NICE-TO-KNOW
• The types of direct examination used in the identification of
fungal infections include wet preparation such as KOH, India
ink, calcofluor white stain, and gram stain
® Although the gram stain performed in routine
microbiology laboratory often gives the first evidence of
infections with bacteria and yeast, other direct stains
give more specific information about mold infections
® Histologic stains for tissue may also be useful
1. KOH PREPARATION
• Drop of 10-20% KOH + sample (skin and nail scrapings, hair,
tissue) on a slide is heated
® 10-20% solution of KOH is useful for detecting fungal
elements embedded within the skin, hair, nails and tissue
® In this procedure, a drop of the KOH preparation is placed
on a slide. Nail scrapings, hair, skin scales or thin slices of
tissue is then added to the drop and a coverslip is placed
® The slide is then gently heated and then allowed to cool for
approximately 15 minutes
• KOH and heat break down/ dissolve/ soften/ digest keratin and
skin layers
® Results to the clearing of the sample and specimen thus
allowing the better visualization of fungal elements
• Modification of KOH test incorporates dimethyl sulfoxide
(DMSO) and a stain into the KOH solution.
® This modification can provide an easier and more reliable
result
® The problem with the non-modified KOH preparation will
take a longer time and the presence of the artifact remains
in the background making the interpretation or reading of
the result may prove to be difficult
® Dimethyl sulfoxide will facilitate more rapid breakdown of
the cellular debris without requiring heat
® The stain is taken up by the fungi, making the fungal
elements more visible on microscopic examination
MV
Figure 4.
2. CALCOFLUOR WHITE STAIN
• Sensitive and reliable method for identifying fungi
® Because it provides a better contrast from the debris, cells
and tissue fragments which are found in the background
® Also give a good definition of the fungal structures
• Traditionally, the KOH preparation has been the recommended
method for direct microscopic examination of specimen
however the calcofluor white stain is now believed to be
superior to KOH method
• The disadvantage of using this stain is that:
® it will require the use of fluorescent microscope
® it is more expensive
® it will require expertise when it comes to the reporting of
the result
• Slides may be observed using fluorescent microscope
NEED-TO-KNOW
• Using this fluorescent microscope, the fungal elements will
fluoresce ranging from apple green or white in color
depending on the combination of filters used in fluorescent
microscope
3. GRAM STAIN
• Fungi are gram-positive
® Routinely performed in microbiology because it gives first
evidence of infection with bacteria and yeast.
® RECALL Gram Stain Reagent (Mnemonics: VIAS):
o Primary stain: Crystal Violet
o Mordant: Gram’s Iodine
o Decolorizer: Alcohol (Ethyl alcohol)
o Counterstain: Safranin
® Gram positive organism stains purple blue or purple
violet.
® Gram negative organism stains pink or red.
® Fungi are gram positive, so they are purple, blue or violet
in color.
• Used to observe yeast and pseudohyphae present in clinical
samples.
4. INDIA INK PREPARATION
• Used to Examine CSF for the presence of Cryptococcus
neoformans.
® A drop of India ink is mixed with a drop of sediment from a
CENTRIFUGED CSF sample. Then it is examined on
HPO.
• Positive (+) yeast cells surrounded by clear area against a black
background are presumptive evidence of C. neoformans.
• It is a negative stain.
Figure 5. India ink preparation
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 2.01 Specimen Collection and Transport

5. HISTOLOGIC STAINS NEED-TO-KNOW

These are histologic stains applied to stains used in mycology: • The advantage of this procedure is that the arrangement of
conidia is retained. Its major disadvantage is the possible or
a. PERIODIC ACID-SCHIFF (PAS) potential contamination of the colony and the preparation is
• Used to demonstrate carbohydrates. It attaches to the only temporary. This tape preparation should be read
polysaccharide of the fungal cell wall and stains the fungi pink. within 30 minutes and then discarded.
b. METHENAMINE SILVER STAIN
• Best stain in detecting fungal elements. It detects fungi in
histologic sections.
c. PAPANICOLAOU STAIN
• Examines secretion from malignant cells.
d. GIEMSA
Figure 7. Cellophane Tape Method – No need for coverslip because the tape
• Used to detect Histoplasma capsulatum in blood and bone serves a coverslip.
marrow specimen.
Table 1. Primary recovery media used for primary isolation of fungi
e. FONTANA-MASSON STAIN
CULTURE MEDIA INDICATIONS FOR USE
• Used for examination of melanin pigment in cell wall of fungi. Saboraud Dextrose Agar Primary recovery of saprobic
6. TEASE MOUNT and pathogenic fungi; pH 5.6
inhibits bacterial growth
• Most common procedure for microscopic examination is Brain Heart Infusion Agar Primary recovery of saprobic
direct mounting of fungal isolate. It can be achieved by and pathogenic fungi
preparing a tease mount.
Dermatophyte Test Medium Primary recovery of
• Fungal culture + drop of lactophenol cotton blue on a slide dermatophytes; recommended
® We use teasing needles to remove a portion of fungal as screening medium
culture. It is then placed on fungal culture or mycelium on
Potato Flake Agar Primary recovery of saprobic
a slide with a drop of lactophenol cotton blue. and pathogenic fungi
• LPCB kills, preserves and stains the organism. Mycosel Agar Primary recovery of
7. SLIDE CULTURE dermatophytes
• Demonstrates natural morphology of fungal structures and Brain Heart Infusion Agar Primary recovery of saprobic
encourages conidiation in some fungi. with antibiotics and pathogenic fungi exclusive
of dermatophytes
® Fungi is grown directly on the slide on a thin film of agar.
Yeast-Extract Phosphate Primary recovery of pathogenic
® It also encourages conidiation which is the growth and
development of conidia in some fungi. Agar fungi exclusive of
dermatophytes
® It is a rapid method which involves growing of fungi directly
Chromogenic agar Isolation and presumptive
on a slide on a thin film of agar.
identification of yeast and
® Slides can be stored indefinitely.
filamentous fungi

NEED-TO-KNOW
• Unlike bacteria, fungi do not have same nutritional
requirements and environmental needs as characterized of
bacteria. There are very few types of culture media for the
primary isolation of fungi and SDA and BHIA are very
common already.
Figure 6. Agar which is cut in a small square for slide culture • Gentamicin and chloramphenicol inhibit bacterial growth.
Whereas cycloheximide inhibits bacteria and at the same
NEED-TO-KNOW time many of the environmental fungi are usually considered
• The purpose of lactophenol cotton blue kills, preserves and as contaminants (also serves as antifungal agent).
stains organism. Lactic acid preserves the organism; • Gentamicin, chloramphenicol and cycloheximide are the
Phenol kills the organism; Cotton blue serves as the dye antimicrobial usually included in fungal culture media.
which stains the organism or chitin in the fungal cell wall.
• Major disadvantage is disruption of conidia because of Table 2. Differential test media
teasing because we use teasing needles. CULTURE MEDIA INDICATIONS FOR USE
Birdseed (Nigerseed) agar For growth of Cryptococcus
8. CELLOPHANE TAPE PREPARATION neoformans
• Involves touching the surface of the colony with a piece of clear Trichophyton Agars Identification of the
tape Trichophyton species
• Tape is placed on a slide with Lactophenol cotton blue and Germ tube media (rabbit, To demonstrate germ tube
examined under the microscope. fetal calf, or human serum) production by the yeast Candida
® The procedure is much the same like cellulose tape albicans
method for the detection of the parasite Enterobius Urea agar slant To demonstrate urease
vermicularis which is also called as pinworm or seat worm. production.
(+) pinkish purple within 48
hours after inoculation.
Cornmeal agar with Tween Identification of Candida
80 and trypan blue albicans by chlamydospore
production; identification of C.

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 2.01 Specimen Collection and Transport

albicans by microscopic • Detection of cryptococcal antigen, in CSF or serum using latex

morphology agglutination assays and enzyme immunoassays (useful in
Czapek’s agar Differential identification of diagnosis of cryptococcosis)
Aspergillus spp. ® Antibody testing – useful but not in immunocompromised
Potato dextrose agar Demonstration of pigment patients who are incapable of producing a measurable
production by Trichophyton humoral response.
rubrum; ® Complement fixation - classic serologic test; another
Rice Medium Identification of Microsporum sensitive method but is difficult to perform and interpret.
audouinii ® Immunodiffusion testing – used to detect antigens of
Cottonseed conversion Conversion of the dimorphic Histoplasma capsulatum; 100% specific but insensitive
agar fungus Blastomyces spp. from and not used as screening tool. This test also requires 2-3
mold to yeast form weeks to exhibit positive result.

Table 2
• Are culture media used for demonstrating definitive/specific
or particular group of fungi. It also used to demonstrate
certain properties such as production of germ tube urease
and pigments.
*Note: Study both tables since they are included in the unit
test :<
B. SPECIAL TESTS
1. GERM TUBE TEST
• The easiest and most important test to identify yeast. It involves
the use of either serum or plasma.
• Differentiates Candida albicans from other yeasts
® (+) Filamentous outgrowth extending from the yeast cells
PROCEDURE
1. Emulsify a yeast colony in 0.5 ml of sheep or human serum in
a small tube. Incubate the tube at 35°C for 3 hours.
2. Transfer a drop of the serum to a slide. Place coverslip and
examine under the microscope.
NEED-TO-KNOW
• Cryptococcal antigen test has been considered as a
primary diagnostic tool in the identification of Cryptococcus
neoformans. This cryptococcal antigen test replaced India
ink preparation in screening the CSF or serum for
Cryptococcus neoformans.
• Cross reaction with other fungal antibodies can also be a
problem.
3. MOLECULAR METHOD
• Polymerase Chain Reaction (PCR) aids in the diagnosis of
microscopy negative fungal infections in conjunction with other
tests.
® PCR aids in diagnosis of fungal infections which tested
negative using direct microscopic examination.
® In research made by Laws et. Al. on the utility of PCR in
the diagnosis of invasive fungal infection, they found out
that PCR has a sensitivity of 97% and specificity of 57.1%
for microscopy negative fungal infection.
® SENSITIVITY: 97%; SPECIFICITY: 57.1%

NOTE
• For red text
® baliktad yung sinabi ni prof sa discussion and sa
recorded lec posted in canvas. (sensitivity: 57.1%;
specificity: 97%)

Figure 8. differentiates C. albicans which shows true germ tubes from the other
yeasts.

NEED-TO-KNOW
• Test is considered positive or there is a presumptive
identification of Candida albicans if you have a presence of
true germ tube. True germ tube lacks constriction (left
photo; figure 8). There is constriction on the base of the
pseudogerm tube (right). True germ tubes lacks
constriction at their bases where they attach to the mother
cell.
• If constriction is present at the base of the germ tube, it is
called pseudo germ tube which are more characteristic of
Candida tropicalis.
2. SEROLOGIC TESTS
• These are available both for the detection of antigen and
antibodies to selected fungal antigens like (refer to the next
bullet)
• Detection of Cryptococcus, Blastomyces, Aspergillus and
Histoplasma.
® The most important application of serologic test is the
detection of cryptococcal antigen in CSF or serum.

MV 6 of 6
 Cellophane Tape & Slide Culture Preparation MYCO&VIRO
Prof. Esmeralda Abling LE 02
31 January 2022 TRANS 02

OUTLINE II. SLIDE CULTURE TECHNIQUE FOR FUNGI

I. Cellophane Tape Preparation • Slide culture method is a rapid method of preparing
II. Slide Culture Preparation fungal colonies for examination and identification.
→ This method permits the fungi to be studied
I. CELLOPHANE TAPE PREPARATION virtually insitu with as little disturbance as
Step 1 Prepare crystal scotch tape forceps and scissors possible.
→ Fungi are identified mostly by close
examination of its morphology and the
characteristics it possess.
→ In slide cultures, we are growing the fungi
directly on the slide on a thin film of agar.
Materials Required:
7-10 days old fungal culture
Saboraud Dextrose Agar plate
U-shaped glass rod placed in a petri plate with a
sterile filter paper with 9cm diameter placed
Step 2 Cut a small piece of scotch tape Microscope Slides and coverslip
Scalpel
Inoculating loop
95% ethanol
Lactophenol cotton blue stain
Sterile Distilled Water

Step 1 Arrange all materials in the laminar chamber.

Step 3 Drop the aniline blue in lactophenol dye in the

center of the slide

Step 2 Aseptically, place the sheet of sterile filter paper in a

petri dish

Step 4 Gently touch the surface of the colony with the

adhesive side of the scotch tape

Step 3 Place the U-shaped glass rod on the filter paper.

Step 5 Put the fungally imprinted side of the tape onto the
drop of the dye

Step 4

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2.01 Cellophane Tape and Slide Culture Preparation

Put sterile distilled water on the filter paper to

completely moisten it.

Step 5 Gently flame the scalpel to sterilize and cut a fine mm

square block of the medium from the plate containing
SDA.

Step 9 Inoculate four sides of agar square with spores or

mycelial fragments of the fungus to be examined.

Step 10 Cover the petri dish and incubate at room

Step 6 Pick up the block of agar by inserting the scalpel and temperature for 48 hrs.
carefully transfer the block aseptically on the center
of the slide.

Step 11 After 48 hours, examine the slide. Mycelial growth

and spore production is observed through the four
Step 7 Asepticaly, place a sterile coverslip on the upper corners the agar cube spreading over to the
surface of the agar cube. coverslip.

Step 12 Procedure for application of LPCB. Place a drop of

Step 8 Flame the inoculation loop until red hot. Take the lactophenol cotton blue stain on a clean microscope
fungal culture and remove a small amount of fungal slide.
culture from the plate.

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2.01 Cellophane Tape and Slide Culture Preparation

Step 13 Remove the cover glass from the slide culture and
discard the block of agar.

Step 14 Place coverglass with mold grown side down, on the

droop of LPCB

Step 15 Examine the slide under low power and then HPO of
the microscope and morphological features of agr
can be observed.

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