Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical
Sciences
Department of Medical Laboratories
STUDENT'S LABORATORY MANUAL
Course code
Course name
MDL 354
Medical Mycology
Course Outcomes
1.1.1. Recognize basic knowledge of medical fungi; their distribution, Morphological characteristics,
pathogenesis/diseases caused, control and means of treatment. @ (1.1).
1.1.2. Describe mold and yeasts @ (1.2).
2.1.1. Explain, summarize, compare, interpret and predict the different aspects of fungi and their infections
in humans. @ (2.1).
2.2.2. Diagnose and evaluate the rate and severity of fungal infections. @ (2.2)
3.2.2. Work effectively in team @ (3.2)
5.1.1. Operate certain equipment’s and instruments to examine and diagnose the different fungi. @ (5.1).
5.2.2. Prepare mycology media for isolation of fungi and examine different microscopic morphology of fungi @ 5.2
Revision No.
Date
Prepared by
Approved by
1
Dr. R. Alarousy
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical
Sciences
Department of Medical Laboratories
Laboratory Safety and General Considerations:
Refer to Laboratory Safety Manual
Note the following when processing Mycology isolates:
ALL work on filamentous fungus is carried out in LAMINAR AIRFLOW BIOSAFETY CABINET TYPE 2. Bio safety Level 2
procedures are recommended for personnel working with clinical specimens that may contain dimorphic fungi as
well as other potential pathogenic fungi. Gloves should be worn for processing specimens and cultures.
If the FILAMENTOUS FUNGUS FORM of a dimorphic fungus is growing or suspected, BIOSAFETY LEVEL 3 procedure
and containment should be followed i.e. wear a N95 mask in addition to what is required for Bio safety Level 2
containment.
Wipe off working area with Virox before and after each day's work. If a culture is dropped or spilled, pour Virox over
the contaminated area, cover with paper towels and let stand for at least 15 minutes. Wipe off the surface and
deposit the contaminated material in an appropriate biohazard disposal container. Clean the surface again using
70% alcohol.
Screening and reading cultures:
2
i)
Sort the plates from sterile specimens and place them into a separate rack.
ii)
Read all cultures (except special requests for dimorphic fungi) daily for the first week and two times a
week (separated by at least one day) for the remaining incubation period. Work up positive specimens
immediately.
iii)
For special requests for dimorphic fungi, read cultures daily for the first two weeks and three times a
week (separated by at least one day) for the remaining 4 weeks of incubation. Work up positive
specimens immediately.
iv)
LPAB (Lactophenol Aniline Blue) preparations are made at least twice a week or daily depending on
volumes. Any mold referred from the bacteriology section is processed and worked up the same day
(except weekends and holidays). All positive LPAB preparations are checked by the other Mycology
technologist or the microbiologist.
1. Result & Interpretation
See the chart for macroscopic morphology of fungi then illustrate your demonstrated colonies.
A. flavus and A. fumigatus
Fusarium spp.
3
Aspergillus fumigatus
Absidia spp.
Candida albicans
Cryptococcus neoformans
Candida kefyr
Histoplasma capsulatum
Zygomycetes


Multiple different genera included
Colony appearance will vary based on genera/species
Microscopic
Appearance:



Broad hyphae, aseptate, or rarely septate
Can include rhizoids and stolons
Sporangiophores are comprised of sporangia on the ends, which
house the sporangiospores
Laboratory
Identification:



Air
Direct/Surface
Bulk
Allergenic Health
Effects:




Common Allergen
Asthma
Hay Fever
Pneumonitis
Colony Morphology
4
Pathogenic Health
Effects:

Yes — immunocompromised individuals (rare occurrence)
Toxins Produced:

Unknown



Grows moderately rapid
Texture — powdery
Pigment — white, pink, orange, black on surface, pale,
orange, pink, black on reverse


Hyphae — septate, hyaline
Conidiophore — hyaline or pigmented, simple or branched,
smooth or rough walls
Phialides hyaline or brown, ellipsoidal, comprised in groups
of 3 — 10 at the tips of conidiophores
Conidia — black single — celled, ellipsoidal, smooth to rough
walled, located in slimy groups at the tips of phialides
Image:
Stachybotrys
Colony Morphology
Microscopic
Appearance:


Laboratory
Identification:
Allergenic Health
Effects:
5



Air
Direct/Surface
Bulk

Unknown
Pathogenic Health
Effects:
Toxins Produced:
Image:
6

Unknown


Satratoxin H
Trichothecene toxins
Penicillium/Aspergillus
Colony Morphology
Microscopic
Appearance:
Laboratory
Identification:
Allergenic Health
Effects:
Pathogenic Health
Effects:
Toxins Produced:
Image:
7



Usually grows moderately rapid/rapidly
Texture — velvet — like/powdery
Pigment — green, blue — green, grey — green, white, yellow
or pinkish on surface, pale/yellowish, sometimes red or brown
on reverse



Hyphae — septate, hyaline
Conidiophores — simple or branched
Phialides are bunched in brush — like clusters (penicillin) at
the tips of conidiophores
Conidia — single — celled, round/ovoid, hyaline or
pigmented, rough or smooth walls in chains




Air
Direct/Surface
Bulk




Common Allergen
Asthma
Hay Fever
Pneumonitis

Yes — immunocompromised individuals (rare occurrence)

Yes — specific to the genera/species
Fusarium
Colony
Morphology
Microscopic
Appearance:
Laboratory
Identification:
Allergenic
Health Effects:
Pathogenic
Health Effects:
Toxins
Produced:
Image:
8



Usually grows rapidly
Texture — wooly/sometimes mucus — like
Pigment — white, yellow, pink, purple, or pale brown on surface,
pale, red, violet, brown, blue on reverse




Hyphae — septate, hyaline
Phialides — long or short, cylindrical, simple or branched
Microconidia — single — celled, sometimes bicellular, hyaline,
ovoid/ellipsoid in chains
Macroconidia — curved, multicellular w/ a foot cell at the base



Air
Direct/Surface
Bulk

Unknown

Yes — reported amongst immunocompromised individuals, as
well as normal hosts





Trichothecene toxins
Vomotoxin
T — 2 toxin
Fumosin
Zearalenone toxin
Aspergillus
Colony
Morphology

Multiple different species included, each with a varying colony
morphology

Conidiophores end with a sac — like structure, phialides are
attached to this sac — like structure (uniserate) or on cells called
metula (biserate), and conidia are attached to phialides in chains



Air
Direct/Surface
Bulk


Allergen
Asthma

Yes — reported amongst immunocompromised individuals, as well
as normal hosts (rare occurrence in normal hosts)


Yes — specific to the species
Examples include aflatoxins and ochratoxin
Microscopic
Appearance:
Laboratory
Identification:
Allergenic
Health Effects:
Pathogenic
Health Effects:
Toxins
Produced:
Image:
9
Ascospores


Multiple different genera included
Colony appearance will vary based on genera/species


Spores are produced in an ascus (sac — like structure)
Multiple spores which vary based on genera/species



Air
Direct/Surface
Bulk
Allergenic Health
Effects:

Common Allergen
Pathogenic Health
Effects:

Potential Health Hazard (rare occurrence)

Yes — specific to the genera/species
Colony Morphology
Microscopic
Appearance:
Laboratory
Identification:
Toxins Produced:
Image:
10
2.
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 05: Microscopically Identification of Yeasts: Wet mount Technique
1. Objectives:
 Fungal cells can be seen easily and clearly, when colored by stains, but in most of the staining processes, the
cells die and lose their natural shape and size due to heat-fixation as well as due to exposure to chemicals
(stains, acid and alcohol).
 The aim of this experiment is to observe the natural shape, size and arrangement of bacteria in living
condition. Motility cannot be observed in wet mount, as the cells are pressed between the slide and the cover
slip.
2. Principle:
In wet mount, a drop of the yeast suspension is placed on a slide, covered with a cover slip and observed
under a light or compound microscope or preferably under a dark-field or phase-contrast microscope using
oil-immersion objective.
3. Materials:
Glass slides.
Cover slides.
Distilled water.
Yeast suspension.
Light microscope.
Cedar oil.
4. Precautions:
Follow Laboratory Safety Precautions.
5. Procedure:
Put one drop of water on the slide. (Using a water dropper)
Place a drop of yeast suspension on the slide. (Using a dropper, Pasteur or loop)
Lower the cover glass slowly to avoid air pockets, pull the tweezers out.
11
After placing the cover glass, the excess water should be absorbed with paper.
Place your prepared wet mount on the “stage” of the microscope.
Examine using oil immersion lens.
6. Result & Interpretation
7. Clinical Significance:
Wet mount technique is simple technique used basically to identify fungal strains and to examine the fine cell
structure of each class (yeasts& molds).
8. References:
Alarousy, R. M. (2004): Molecular Biology Studies on Cryptococcus neoformans. Master Degree Thesis, Cairo
University.
12
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 06: Microscopically Identification of Yeasts: Germ Tube Test.
1. Objectives:
-
The main objective is to learn how to apply rapid laboratory tests to differentiate between different species
of Candida.
2. Principle:
The Germ Tube Test is a screening procedure used to differentiate Candida albicans from other yeast. Approximately
95 - 97% of Candida albicans isolated develop germ tubes when incubated in a proteinaceous media.
Germ tubes are short non-septate germinating hyphae. They are ½ the width and 3 - 4 times the length of the cell
from which they arise. The junction of the germ tube and cell is not constricted. Buds and pseudo-hyphae can be
distinguished from germ tubes by the constricted attachment.
3. Materials:
 Bovine serum - A small volume to be used as a working solution may be stored at 2 to 8oC. Stock solution
can be dispensed into small tubes and stored at -20oC.
 Clean glass microscope slides
 Glass coverslips
 Glass tubes (13 x 100 mm)
 Pasteur pipettes.
 Cell suspension of C. albicans.
4. Precautions:
 General laboratory precautions.
5. Procedure:
 Put 3 drops of serum into a small glass tube.
 Using a Pasteur pipette, touch a colony of yeast and gently emulsify it in the serum. The pipette can be left
in the tube.
 Incubate at 35oC to 37oC for up to 3 hours but no longer.
 Transfer a drop of the serum to a slide for examination.
 Coverslip and examine microscopically using x 40 objective.
6. Result & Interpretation
13
Positive test: presence of short lateral filaments (germ tubes) one piece structure
Negative test: yeast cells only (or with pseudohyphae) always two pieces
Positive germ tube (C. albicans)
Please add your lab. picture:
7. Clinical Significance:
Germ tube test is used for identification of positive germ tube – Inducing yeasts (C. albicans).
8. References:
www.doctorfungus.org.
Shaimaa Y. Aboulmagd (2011): MD Thesis”Some Mycological and Molecular biological Studies on Mixed Yeast
Infection’’.
14
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 07: Microscopically Identification of Yeasts: Indi Ink for observation of Capsule.
1. Objectives:
 The test aim is to learn an easy and rapid technique differentiates Cryptococcal strains.
2. Principle:
 The procedure is applicable only to suspected positive Crytococcal cultures.
 The mucoid capsule appears as a clear halo that surrounds the yeast cell or lies between the cell wall and the
surrounding black mass of India ink particles. Capsules may be broad or narrow. The yeast cells may be
round, oval or elongate. Buds may be absent, single or rarely multiple and may be detached from the
mother cell but enclosed in a common capsule attached.
3. Materials:
Glass slides& coverslips.
Cedar Oil
India ink stain.
Cell suspension from C. neoformans culture.
Light microscope
4. Precautions:
General Laboratory safety precautions.
Great care of infection with C. neoformans
5. Procedure:
 For suspected Crytococcal cultures, make a wet preparation using saline on a clean glass slide, then add a
small drop of India Ink and mix.
 Apply a large coverslip ((22 x 40 mm) over the mixture and press it gently to obtain a thin mount.
 If India Ink is too thick (dark), dilute it by 50% with saline.
 Allow the preparation to stand for few minutes to settle.
 Scan under low power in reduced light. Switch to high power if necessary.
15
6. Result & Interpretation
India Ink preparation:
Encapsulated yeasts (400x)
Please add your lab. picture:
7. Clinical Significance:
Capsule of encapsulated yeasts (as Cryptococcus neoformans) can be detected using India ink stain. Thus we can
use this technique for confirming the presence of this yeast directly in the samples.
8. References:
 Murray PA, et al. Manual of Clinical Microbiology 7th ed., 1999, ASM Press.
 Alarousy, R. M. (2004): Molecular Biology Studies on Cryptococcus neoformans. Master Degree Thesis, Cairo
University.
16
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 08: Macroscopic Identification of Molds “Comparing Different Species of Molds”
1. Objectives:
By the end of this section, students should be able to, primarily, identify molds macroscopically using Atlas or available
charts.
2. Principle:



The majority of filamentous fungi recovered in the clinical laboratory belong to the class Hyphomycetes, which
includes all of the filamentous, sporulating members of the Fungi Imperfecti .
Zygomycetes, Ascomycetes and Basidiomycetes produce characteristic spores after the processes of plasmogamy,
karyogamy, and meiosis; therefore, zygospores, ascospores, and basidiospores are not commonly seen in the
clinical laboratory because only one mating type is usually recovered.
Many criteria are considered when identifying moulds. Morphology, culture characteristics, temperature
tolerance, cycloheximide resistance, dimorphism, nutritional requirements, proteolytic activity, and the ability to
hydrolyze urea are criteria used in identifying moulds. Modern classification schemes primarily emphasize
conidial ontogeny rather than color, conidial septation or the appearance of growth on natural substrates.
3. Materials:
Macroscopic examination will be done using naked eyes.
4. Precautions:
General laboratory and safety precautions.
Avoid opening the plates
5. Procedure:
Macroscopic Examination:




17
Colonial morphology
Surface pigment on non-blood containing medium
Reverse pigment on non-blood containing medium
Growth on cycloheximide containing medium.
I.
procedure of reading of cultures
Specimen
Incubation Period at 28oC
Comment
All specimens other than
special requests for dimorphic
fungi, Malassezia or
environmental specimens
4 weeks
Read daily for 1st week; then 2 times
per week for the remaining 3 weeks.
Special Request Dimorphic
6 weeks or Send to PHL
Read daily for 2 weeks; then 3 times
per week for the remaining 4 weeks.
Special Request Malassezia
1 week
Read daily for 1 week
Environmental
7 days
Read on Day 1 and then on Day 5.
ii.
Identification
a)
Filamentous fungi
Most filamentous fungi can be identified based on a combination of colonial morphology and microscopic features.
Pathogenic dimorphic fungi such as Blastomyces, Histoplasma, Sporothrix, etc., can often be presumptively identified
by the presence of their characteristic conidia seen on Lactophenol Aniline Blue (LPAB) preparations of culture
isolates.The extent to which a filamentous fungus is identified in the laboratory will depend on several factors. The
following should be used as a guide. If there is any question regarding the extent to which a filamentous fungus
should be identified, consult with the microbiologist or senior mycology technologist.
a) Sterile site specimens:
Identify all filamentous fungi isolated. Possible culture contaminants (e.g. a single colony of Penicillium
species or other saprophytes growing on only one of several media) should be checked with the Charge
Technologist or the Microbiologist before proceeding.
b) All other specimens:
Identify all filamentous fungi isolated.
Procedure:
Examine the culture plates as per Reading of Cultures Schedule and record the macroscopic and microscopic
findings in the LIS Media Comment field.
Macroscopic Examination:
c)
d)
e)
f)
Colonial morphology
Surface pigment on non-blood containing medium
Reverse pigment on non-blood containing medium
Growth on cycloheximide containing medium

Growth at 37oC

Refer to the experiment of 4th week.
With the help of teacher:
18




Pathology report if available
Clinical data
See the Charge Technologist or Microbiologist for consultation if needed.
Send isolate to the Public Health Lab if significance/identification cannot be determined
immediately.
g) If the filamentous fungus does not produce conidia, subculture the fungus onto the media as outlined
below. Re-incubate the original plates for the remaining incubation period.
Media
Incubation
Coloured Mould:
i)
ii)
iii)
Potato Dextrose Agar (PDA)
O2,
28oC
SAB
O2,
37C
Examine the sub-cultured plates daily and record findings in the LIS Media Comment field.
If there is no growth after 7 days, forward the original culture plate to the Public Health
Laboratory (PHL) for further work-up.
When sufficient growth is noted, record:
Macroscopic Examination:
a) Colonial morphology
b) Surface pigment
c) Reverse pigment
d) Growth on cycloheximide containing agar
Microscopic Examination:
a) Prepare LPAB preparation(s) from subculture plates as required depending on colonial
morphology on each plate and examine under light microscope as outlined above.
b) If there is growth without conidia production and growth on SAB 37C plate, send the isolate
to PHL for further work-up.
19
c) If there is growth without conidia production and no growth on SAB 37C plate, determine
the significance of the isolate by taking into consideration the following:
 Direct smear result
 Growth on cycloheximide containing media
 Refer to the FLOW CHARTS for IDENTIFICATION
With the help of on-call resident or microbiologist:
 Pathology report if available
 Clinical data
 See the Charge Technologist or Microbiologist for consultation if needed.
 Send the isolate to PHL for further work-up if needed.
d) Send the isolate to PHL for further work-up.
e) If there is growth with conidia and the isolate cannot be identified, set up a slide culture (see
Appendix VI - Slide Culture). If the isolate cannot be identified by slide culture, send the
isolate to PHL for further work-up.
For white filamentous fungus:
1. Re-incubate the culture for another 48 hours. If the fungus remains white after 48 hours, seal and send
the culture to the Public Health Laboratory for identification. DO NOT manipulate the culture. If the
fungus becomes a coloured mould after 48 hours, prepare a tease mount or scotch tape preparation of
each fungus colony type from each media using Lactophenol Aniline Blue (LPAB).
2. Report the identification according the instructions in the Reporting Section in the 4th week above.
6. Result & Interpretation
Refer to the experiment of 4th week above.
20
7. Clinical Significance:
Although it is considered an important assay for identifying moulds, but characteristics which vary depending on the
media and environmental conditions, or which are subjective, such as color are not consistent enough to be used as the
sole criteria in classifying moulds.
8. References:
UNIVERSITY HEALTH NETWORK /Mount Sinai HOSPITAL, DEPARTMENT OF MICROBIOLOGY
Quality Manual. Subject Title: Pre-analytical Procedure - Specimen Collection. Approved by Lab. Director
21
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 09: Mold Identification Techniques: Adhesive Tape Technique
1. Objectives:
Identify fungal species based on a combination of colonial morphology and microscopic features using simple
technique.
2. Principle:
 Most filamentous fungi can be identified based on a combination of colonial morphology and microscopic
features.
 Pathogenic dimorphic fungi such as Blastomyces, Histoplasma, Sporothrix, etc., can often be presumptively
identified by the presence of their characteristic conidia seen on Lactophenol Aniline Blue (LPAB)
preparations of culture isolates. The extent to which a filamentous fungus is identified in the laboratory will
depend on several factors. The following should be used as a guide. If there is any question regarding the
extent to which a filamentous fungus should be identified, consult with the microbiologist or senior
mycology technologist.
a) Sterile site specimens:
Identify all filamentous fungi isolated. Possible culture contaminants (e.g. a single colony of Penicillium
species or other saprophytes growing on only one of several media) should be checked with the Charge
Technologist or the Microbiologist before proceeding.
b) All other specimens:
Identify all filamentous fungi isolated.
3. Materials:
Cultures of filamentous fungi (molds).
Glass slides
Coverslips.
Lactophenol cotton blue.
Vasline
Scotch tape.
Light microscope.
4. Precautions:
General laboratory and safety measures.
22
5. Procedure:
Using ungloved hands begin in preparing the slide:
1.
2.
Place a drop of LPAB onto a clean glass slide, make a circle with Vaseline.
Take a small piece of clear scotch tape and loop back on itself with sticky sides out.
3.
Hold the tip of the loop securely with forceps.
4.
Press the sticky side firmly to the surface of the fungal colony.
5.
Pull the tape gently away from the colony.
6.
Open up the tape strip and place it on the drop of LPAB on the glass slide, making sure that the
entire sticky side adheres to the slide. Add cover slip.
7.
Examine under the light microscope.
Microscopic Examination:
1. Under the light microscope, examine the slide(s) for the presence, shape, size and attachment of
conidia. Compare and match the above features with those described in a reference textbook.
2. If the filamentous fungus can be identified from the LPAB preparation, mark the identified colony (ies)
with an “X” on the back of the culture plate(s) [if more than one type of fungus is identified, place
number (e.g. 1, 2, 3, etc) beside the “X” which matches the number and identification entered into the
LIS]. Re-incubate the original culture plates for the remaining incubation period and examine plates for
23
additional growth. Hold at room temperature, if plates completely over grown by 4 weeks; discontinue
incubation; and report the identification.
3. Set up a slide culture if unidentified due to overlapping conidial structures with other species.
4. If the filamentous fungus is producing conidia but cannot be identified, determine the significance of the
isolate whether it is a probable pathogen, a possible pathogen (i.e. opportunistic fungus) or an unlikely
pathogen (i.e. saprophyte), take into consideration the following:

Direct smear result

Growth on cycloheximide containing media.
Student’s notes:
24
6. Result & Interpretation
7. Clinical Significance:
Identify all filamentous fungi isolated.
8. References:
Davise H. Larone. Medically Important Fungi. A Guide to Identification. 3rd ed. 1995, ASM Press.
Martha E. Kern. Medical Mycology. 1985.
25
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 10: Mold Identification Techniques: Slide Culture Technique
1. Objectives:
The students will be trained on preparing the slide culture technique and examine the slides under light microscope
to identify filamentous fungi.
2. Principle:
LPAB contains lactic acid as a clearing agent, phenol as a disinfectant, glycerol to prevent drying and Aniline Blue
which is the dye that stains fungi. LPAB is a wet preparation.
3. Materials:











LPAB reagent
Mold culture
Probe to get the specimen
Teasing needles
Glass slides
Coverslips
Lead or wax pencils
Disinfectant bucket
Electric incinerator
Clear nail polish
Slide tray
4. Precautions:
General laboratory safety precautions.
5. Procedure:
The test must be performed in the Laminar Airflow Biosafety Cabinet. First, observe the gross morphology of the
colony carefully to determine whether or not the culture is mouldy, granular or a mixture of both. It is important to
prepare the LPAB preparation by "teasing" the fungus not "chopping" it.
26
One plate of nutrient agar; potato dextrose or Sabouraud’s Dextrose Agar (SDA) is recommended, however,
some fastidious fungi may require harsher media to induce sporulation like cornmeal agar or Czapek dox
agar.
1. Using a sterile blade cut out an agar block (7 x 7 mm) small enough to fit under a coverslip.
2. Flip the block up onto the surface of the agar plate.
3. Inoculate the four sides of the agar block with spores or mycelial fragments of the fungus to be grown.
4. Place a flamed coverslip centrally upon the agar block.
5. Incubate the plate at 26C until growth and sporulation have occurred.
6. Remove the cover slip from the agar block.
7. Apply a drop of 95% alcohol as a wetting agent as water.
8. Gently lower the coverslip onto a small drop of Lactophenol cotton blue on a clean glass slide.
9. The slide can be left overnight to dry and later sealed with fingernail polish.
10. When sealing with nail polish use a coat of clear polish followed by one coat of red coloured polish.
https://www.youtube.com/watch?v=FcW5tHrbAos
6. Result & Interpretation
27
7. Clinical Significance:
To determine the morphology of the conidiogenous cells and the conidia that they give rise to in order to identify a
filamentous fungus.
8. References:
Clin. Micro. Newsletter 9:33-36, Mar. 1, 1987. K.L. McGowan. "Practical Approaches to Diagnosing Fungal Infections
in Immunocompromised Patients".
28
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 11: Full Identification of Filamentous Fungi (Quiz)
1. Objectives:
The objective of this section is to learn the full protocol of identifying filamentous fungi.
2. Principle:
Using combination between colonial morphology (macroscopic) and microscopic morphology is very useful in identifying
filamentous fungi.
3. Materials:

29
Will be written by the student:
4. Precautions:

General laboratory safety precautions.
5. Procedure:
Report the identification according the instructions in the reporting section:
6. Result & Interpretation
REPORTING
1.
FUNGAL STAIN:
Negative Reports: "No Fungal elements seen".
Positive Reports:




Fungal elements seen.
Yeast seen (with quantitation)
Yeast with pseudohyphae seen (with quantitation)
Filamentous fungus seen (without quantitation); with morphologic description of
organisms/structures seen (e.g. septate hyphae). Define structures.
If unable to interpret fungal elements, consult Senior Mycology Technologist.
30
2.
CULTURE:
Negative Reports: "No Fungus isolated".
Positive Reports:
 When reporting a fungus culture result, DO NOT quantitate filamentous fungi.
7. Clinical Significance:
8. References:
31
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 12: The Diphasic Fungi
1. Objectives:
Histoplasmosis is the most famous diphasic (dimorphic) fungi and identification of its two phases (yeast& mold
phases) is very important.
2. Principle:
H. capsulatum exhibits thermal dimorphism by growing in living tissue or in culture on enriched medium as Brain
heart infusion agar plates at 370C as a budding yeast-like fungus or in soil or ordinary culture medium at
temperatures below 300C as a mould.
3. Materials:
Sabouraud’s dextrose agar plates.
Brain Heart Infusion agar plates.
Suspected colonies.
Platinum loops.
Incubator 370C.
Incubator 300C.
Light microscope.
4. Precautions:
Cultures of H. capsulatum represent a severe biohazard to laboratory personnel and must be handled with extreme
caution in an appropriate pathogen handling cabinet.
5. Procedure:
Cultures that are suspected to be H. capsulatum should be sub-cultured on:
o Sabouraud’s Dextrose Agar medium plate (SDA) and incubated at 250C.
o Brain Heart Infusion Agar (BHIA) and incubated at 370C.
Examine both plates for colonial morphology and microscopic morphology.
6. Result & Interpretation
On Sabouraud's dextrose agar at 25 0C, colonies are slow growing, white or buff-brown, suede-like to
cottony with a pale yellow-brown reverse. Other colony types are glabrous or verrucose, and a red
pigmented strain has been noted. Microscopic morphology shows the presence of characteristic large (8-14
32
um in diameter), rounded, single-celled, tuberculate macroconidia formed on short, hyaline, undifferentiated
conidiophores. Microconidia, if present, are small (2-4 um in diameter), round to pyriform and borne on
short branches or directly on the sides of the hyphae.
On brain heart infusion (BHI) blood agar incubated at 370C, colonies are smooth, moist, white and yeastlike. Microscopically, numerous small round to oval budding yeast-like cells, 3-4 x 2-3 um in size are
observed.
Large, rounded, single-celled, tuberculate macroconidia and small microconidia of H. capsulatum.
33
7. Clinical Significance:
Histoplasmosis is an intracellular mycotic infection of the reticuloendothelial system caused by the inhalation of
conidia from the fungus Histoplasma capsulatum. Approximately 95% of cases of histoplasmosis are inapparent,
subclinical or benign. Five percent of the cases have chronic progressive lung disease, chronic cutaneous or systemic
disease or an acute fulminating fatal systemic disease. All stages of this disease may mimic tuberculosis.
8. References:
Rippon, J.W. 1988. Medical Mycology. 3rd Edition. W.B. Saunders Co., Philadelphia, USA.
34
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 13: Antifungal Susceptibility Testing of Fungi (yeasts)
1. Objectives:
By the end of this experiment, student will be able to prepare yeast inoculum, media and solutions required for
antifungal sensitivity test.
2. Principle:
Dilution methods are used to establish the minimum inhibitory concentrations (MICs) of antimicrobial agents.
Dilution methods are the reference methods for antimicrobial susceptibility testing, and are mainly used to establish
the activity of a new antifungal agent, to confirm the susceptibility of organisms that give equivocal results in routine
tests, and to determine the susceptibility of fungi where routine tests may be unreliable. Fungi are tested for their
ability to grow in microdilution plate wells containing broth culture media and serial dilutions of the antimicrobial
agents (broth microdilution). The MIC is defined as the lowest concentration (in mg/L) of an antifungal agent that
inhibits fungal growth. The MIC provides information concerning the susceptibility or resistance of the organism to
the antifungal agent and can help in making correct treatment decisions.
3. Materials:
RPMI 1640 buffered, pH 7.0
Yeast cells suspensions [0.5 McFarland (1 x 103 to 5 x 103 CFU/ml)].
Antifungal drug solutions.
Micro dilution plates
micro dilution plates reader.
4. Precautions:
General laboratory and safety measures.
5. Procedure:
The test is performed in microdilution plates. The method is based on the preparation of antifungal agent working
solutions in 100 μL volumes per well (with the addition of an inoculum also in a volume of 100 μL).
Medium
35
A completely synthetic medium, RPMI-1640 with glutamine and supplemented with glucose to a final concentration
of 20 g/L (2%), is used.
Preparation of stock solutions
Antifungal drug solutions must be prepared after taking the potency of the lot of antifungal drug powder into
account. The amount of powder or diluent required to prepare a standard solution. Preparation of working solutions
The range of concentrations tested will depend on the organism and the antifungal drug being tested. A two-fold
dilution series based on 1 mg/L is prepared in double strength RPMI 2% G. The RPMI 2% G medium used in the
plates is prepared at double the final strength to allow for a 50% dilution following addition of the inoculum
prepared in distilled water.
Preparation of microdilution plates
Use sterile plastic, disposable, 96 well microdilution plates with flat-bottom wells that have a nominal capacity of
approximately 300 μL. For the wells in each column, from 1 to 10, of the microdilution plate dispense 100 μL from
each of the tubes containing the corresponding concentration (2 x final concentration) of antifungal agent. For
example, with itraconazole, voriconazole or posaconazole, dispense to column 1 the medium containing 16 mg/L, to
column 2 the medium containing 8 mg/L, and so on to column 10 for the medium containing 0.03 mg/L.
To each well of column 11 and 12 dispense 100 μL of double strength RPMI 2% G medium. Thus, each well in
columns 1 to 10 will contain 100 μL of twice the final antifungal drug concentrations in double strength RPMI 2% G
medium with 1% solvent.
Preparation of inoculum
Standardisation of the inoculum is essential for accurate and reproducible antifungal susceptibility tests. The
inoculum should be prepared by suspending five representative colonies, obtained from an 18-24 h culture on
nutritive agar medium, in sterile distilled water. The final inoculum must be between 0.5 x 105 and 2.5 x 105 CFU/mL.
Colony suspension method
1. Culture all yeasts in ambient air at 35 ±2ºC on non-selective nutritive agar medium (Sabouraud’s dextrose agar or
potato dextrose agar) for 18-48 h before testing.
2. Prepare the inoculum by suspending five distinct colonies, ≥1 mm diameter from 24h cultures, in at least 3 mL of
sterile distilled water.
3. Evenly suspend the inoculum by vigorous shaking on a vortex mixer for 15 s. Adjust the cell density to the density
of a 0.5 McFarland standard (Table 5) by measuring absorbance in a spectrophotometer at a wavelength of 530 nm
and adding sterile distilled water as required. This will give a yeast suspension of 1-5 x 106 CFU/mL. Prepare a
working suspension by a 1 in 10 dilution of the standardised suspension in sterile distilled water to yield 1-5 x 105
CFU/mL.
Inoculation of microdilution plates
The microdilution plates should be inoculated within 30 min of preparing the inoculum suspension, in order to
maintain the viable cell concentration. Inoculate each well of a microdilution tray with 100 μL of the 1-5 x 105
CFU/mL yeast suspension. Also, inoculate the growth control wells (column 11), containing 100 μL of sterile drugfree medium, with 100 μL of the same inoculum suspension. Fill column 12 of the microdilution plate with 100 μL of
sterile distilled water from the lot used to prepare the inoculum as a sterility control for medium and distilled water
(drug-free medium only). Incubation of microdilution plates
36
Incubate microdilution plates without agitation at 35 ± 2ºC in ambient air for 24 ± 2 h. An absorbance of ≤ 0.2
indicates poor growth and is most commonly seen amongst strains of Candida parapsilosis and Candida
guilliermondii. Such plates should be re-incubated a further 12-24 h and then re-read. Failure to reach an absorbance
of 0.2 after 48 h constitutes a failed test. As described above, an absorbance of ≤ 0.2 after 48 h for Cryptococcus spp.
should prompt a repeat test with incubation at 30°C.
Reading results
The microdilution plates must be read with a microdilution plate reader. The recommended wavelength for
measuring the absorbance of the plate is 530 nm, although others can be used e.g. 405 nm or 450 nm.
https://www.youtube.com/watch?v=-63hMAX2UEA
6. Result & Interpretation
7. Clinical Significance:
Antifungal susceptibility testing is used to predict therapeutic outcome.
8. References:
 Antifungal Susceptibility Testing: Practical Aspects and Current Challenges
John H. Rex, Michael A. Pfaller,Thomas J. Walsh, Vishnu Chaturvedi, Ana Espinel-Ingroff,Mahmoud A. Ghannoum,
Linda L. Gosey,Frank C. Odds, Michael G. Rinaldi, Daniel J. Sheehan, and David W. Warnock.
 EUCAST DEFINITIVE DOCUMENT EDef 7.2 Revision
37
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 14: Antifungal Susceptibility Testing of Fungi (moulds)
1. Objectives:
By the end of this experiment, student will be able to prepare yeast inoculum, media and solutions required for
antifungal sensitivity test.
2. Principle:
Dilution methods are used to establish the minimum inhibitory concentrations (MICs) of antimicrobial agents.
Dilution methods are the reference methods for antimicrobial susceptibility testing, and are mainly used to establish
the activity of a new antifungal agent, to confirm the susceptibility of organisms that give equivocal results in routine
tests, and to determine the susceptibility of fungi where routine tests may be unreliable. Fungi are tested for their
ability to grow in microdilution plate wells containing broth culture media and serial dilutions of the antimicrobial
agents (broth microdilution). The MIC is defined as the lowest concentration (in mg/L) of an antifungal agent that
inhibits fungal growth. The MIC provides information concerning the susceptibility or resistance of the organism to
the antifungal agent and can help in making correct treatment decisions.
3. Materials:
RPMI 1640 buffered, pH 7.0
Moulds (spores) cells suspensions.
Antifungal drug solutions.
Micro dilution plates
micro dilution plates reader.
4. Precautions:
General laboratory and safety measures.
5. Procedure:
The test is performed in microdilution plates. The method is based on the preparation of antifungal agent working
solutions in 100 μL volumes per well (with the addition of an inoculum also in a volume of 100 μL).
Medium
38
A completely synthetic medium, RPMI-1640 with glutamine and supplemented with glucose to a final concentration
of 20 g/L (2%), is used.
Preparation of stock solutions
Antifungal drug solutions must be prepared after taking the potency of the lot of antifungal drug powder into
account. The amount of powder or diluent required to prepare a standard solution. Preparation of working solutions
The range of concentrations tested will depend on the organism and the antifungal drug being tested. A two-fold
dilution series based on 1 mg/L is prepared in double strength RPMI 2% G. The RPMI 2% G medium used in the
plates is prepared at double the final strength to allow for a 50% dilution following addition of the inoculum
prepared in distilled water.
Preparation of microdilution plates
Use sterile plastic, disposable, 96 well microdilution plates with flat-bottom wells that have a nominal capacity of
approximately 300 μL. For the wells in each column, from 1 to 10, of the microdilution plate dispense 100 μL from
each of the tubes containing the corresponding concentration (2 x final concentration) of antifungal agent. For
example, with itraconazole, voriconazole or posaconazole, dispense to column 1 the medium containing 16 mg/L, to
column 2 the medium containing 8 mg/L, and so on to column 10 for the medium containing 0.03 mg/L.
To each well of column 11 and 12 dispense 100 μL of double strength RPMI 2% G medium. Thus, each well in
columns 1 to 10 will contain 100 μL of twice the final antifungal drug concentrations in double strength RPMI 2% G
medium with 1% solvent.
Preparation of inoculum
Standardisation of the inoculum is essential for accurate and reproducible antifungal susceptibility tests. The final
inoculum must be between 1x105 cfu/mL and 2.5x105 cfu/mL.
Spore suspension method
Subculture the isolates on potato dextrose agar slants, or other culture media on which the fungus is able to
sporulate readily, and incubate at 35°C. Prepare inoculum suspensions from fresh, mature (2-5 day old) cultures.
With some isolates an extended incubation is required for adequate sporulation.
Cover colonies with approximately 5 ml of sterile water supplemented with 0.1% Tween 20. Then carefully rub the
conidia with a sterile cotton swab and transfer them with a pipette to a sterile tube. Homogenize the suspension for
15 seconds with a gyratory vortex mixer at 2,000 rpm. Make appropriate dilutions in order to attain the right
concentration for counting in a haemocytometer chamber. Inoculum preparations should also be examined for the
presence of hyphae or clumps. If a significant number of hyphae is detected (> 5% of fungal structures), transfer 5
mL of the suspension to a sterile syringe attached to a sterile filter with a pore diameter of 11 μm, filter and collect in
a sterile tube. This step, which is seldom needed for Aspergillus spp., removes hyphae and yields a suspension
composed of spores. If clumps are detected, the inoculum is shaken again in a vortexmixer for further 15 seconds.
Repeat this step as many times as necessary, until clumps are no longer encountered. Adjust the suspension with
sterile distilled water to a concentration of 2-5x106 cfu/mL by counting the conidia in a haemocytometer chamber.
Then dilute the suspension 1:10 with sterile distilled water to obtain a final working inoculum of 2–5 x105 cfu/mL
Inoculation of micro dilution plates
The micro dilution plates should be inoculated within 30 min of preparing the inoculum suspension, in order to
maintain the viable cell concentration. Inoculate each well of a microdilution tray with 100 μL of the 1-5 x 105
CFU/mL yeast suspension. Also, inoculate the growth control wells (column 11), containing 100 μL of sterile drug39
free medium, with 100 μL of the same inoculum suspension. Fill column 12 of the microdilution plate with 100 μL of
sterile distilled water from the lot used to prepare the inoculum as a sterility control for medium and distilled water
(drug-free medium only). Incubation of microdilution plates
Incubate microdilution plates without agitation at 35 ± 2ºC in ambient air for 24 ± 2 h. An absorbance of ≤ 0.2
indicates poor growth and is most commonly seen amongst strains of Candida parapsilosis and Candida
guilliermondii. Such plates should be re-incubated a further 12-24 h and then re-read. Failure to reach an absorbance
of 0.2 after 48 h constitutes a failed test. As described above, an absorbance of ≤ 0.2 after 48 h for Cryptococcus spp.
should prompt a repeat test with incubation at 30°C.
Reading results
The microdilution plates must be read with a microdilution plate reader. The recommended wavelength for
measuring the absorbance of the plate is 530 nm, although others can be used e.g. 405 nm or 450 nm.
https://www.youtube.com/watch?v=-63hMAX2UEA
6. Result & Interpretation
7. Clinical Significance:
Antifungal susceptibility testing is used to predict therapeutic outcome.
8. References:
 Antifungal Susceptibility Testing: Practical Aspects and Current Challenges
John H. Rex, Michael A. Pfaller,Thomas J. Walsh, Vishnu Chaturvedi, Ana Espinel-Ingroff,Mahmoud A. Ghannoum,
Linda L. Gosey,Frank C. Odds, Michael G. Rinaldi, Daniel J. Sheehan, and David W. Warnock.
 EUCAST DEFINITIVE DOCUMENT EDef 7.2 Revision
40
Kingdom of Saudi Arabia
Ministry of Higher Education
Majmaah University
College of Applied Medical Sciences
Department of Medical Laboratories
Section No.:
Student Name:
Student ID No. :
W – 15: Revision& Tests:
1. Objectives:
The objective is to evaluate the student’s accommodation for all the previous information.
2. Principle:
3. Materials:
4. Precautions:
41
5. Procedure:
6. Result & Interpretation
42
7. Clinical Significance:
8. References:
43
CALCOFLUOR WHITE STAIN
Purpose
Calcofluor White stain is useful for staining skin scrapings, hair, nail and thick tissue specimens. Calcofluor staining
requires the addition of KOH which helps to dissolve keratinized particles and helps to emulsify solid, viscous
material that may mask the fungal elements. Calcofluor method is NOT suitable for the detection of Pneumocystis
carinii. Calcofluor stained smears are read under the UV microscope as for the Fungi-Fluor stain.
Procedure
1.
Place a portion of the specimen on the slide (select a purulent area if secretions).
2.
Add 1 to 2 drops KOH and emulsify specimen. If it does not clear rapidly, place slide in petri dish and allow
standing about 10 minutes.
3.
If tissue or scrapings, place the slide on 35oC bench top heating block for 15-20 minutes to speed clearing.
4.
Add 1 or 2 drops of calcofluor white reagent and mix thoroughly. Calcofluor white may be added right after
KOH.
5.
Apply coverslip gently. Make sure specimen does not overflow.
6.
Examine under fluorescent microscope (see Fungi-Fluor stain).
7.
Always include a control slide positive for yeast or filamentous fungus.
NB: In the event when no Fungal Stain smear is available, Gram smear may be retrieved for over staining by
Calcofluor White or KOH.
Interpretation
Fungal cell walls fluoresce apple green (see Fungi-Fluor stain).
44
References
1.
Manufacturers' Instructions:
Calcofluor White Reagent - Difco.
2.
45
Clin. Micro. Newsletter 9:33-36, Mar. 1, 1987. K.L. McGowan. "Practical Approaches to Diagnosing Fungal
Infections in Immunocompromised Patients".
APPENDIX IX - MEDIA / REAGENTS
LACTOPHENOL ANILINE BLUE STAIN (LPAB)
Distilled water
20.00 ml.
Lactic acid
20.00 ml.
Phenol crystals
20.00 gm.
Aniline blue
00.05 gm.
Glycerol
40.00 ml.
Dissolve phenol in the lactic acid, glycerol, and water by gently heating. Then add aniline blue.
Purpose: Used for wet mount preparations of fungal cultures
POTATO DEXTROSE AGAR (PDA)
Purpose
Sporulation medium for fungi (can also be used in slide culture).
Slopes:
Potato dextrose agar (CM 139)
Distilled Water
46
39 g.
1000 ml.
Mix well, bring to a boil to dissolve. Cool to 500C (check pH with ATC - pH ± 5.6). Dispense 10 ml amounts
into pre-sterilised UGB bottles. Autoclave (with loose caps) at 1180C/10 minutes. Cool in a slanted position.
Tighten the caps. Label the bottles "PDA". Store at 40C.
Plates:
Potato dextrose agar
Dist. H2O
39.0 g.
1000.0 ml.
Mix well, bring to a boil. Cool to 50oC (check pH with ATC - pH ± 5.6). Autoclave at 121oC/15 minutes. Cool.
Pour plates (label "PDA"). Shrink wrap plates individually. Store at 4oC.
Quality Control:
Organisms
Incubation
Results
Trichophyton mentagrophytes ATCC 9533
28oC (1 - 3 days)
Good Growth
SABOURAUD AGAR MODIFIED (DIFCO)
Purpose
To provide a better and less inhibitory medium than the original formula for isolation and subculture of
fungi.
Principle
The modified Sabouraud contains 2% rather than 4% dextrose and has a near neutral pH i.e. 7.0 as compared
to 5.6. Other antibiotic containing media can now replace the very low pH in the original formula for
suppressing bacterial contaminants.
Sabouraud Agar (Modified)
50 g.
Distilled Water
1000 ml.
Mix well pH 7.5
Heat to boiling to dissolve.
47
For slopes:
Dispense 10 ml. amounts into UGB bottles. Autoclave 121oC/15 minutes. Slope.
Store at RT. Final pH 7.0 at 25oC.
For plates:
Autoclave flask 121oC/15 min. Cool. Pour plates. Store in plastic bags at 4oC.
Formula per litre
Bacto-neopeptone
10 g.
Bacto-Dextrose
20 g.
Bacto-Agar
20 g.
Quality Control
Organisms
Incubation
Results
Trichophyton mentagrophytes ATCC 9533
25oC
Good Growth
Candida albicans ATCC 10231
25oC
Good Growth
Escherichia coli ATCC 25922
25oC
Inhibited
Best Wishes
Dr. Randa M. I. Alarousy
48