Laboratory Manual
for
Medical Mycology
Based on a Lab Manual by Dr. Allen C. Nelson,
Professor at University of Wisconsin-La Crosse 1966-1996
Modified by Tom Volk Spring 2011
BIO 413/513
Preface
The purpose of this manual is to present laboratory techniques for the identification of
fungi causing mycotic and actinomycotic diseases in humans. The first three exercises are an
introduction to the fungi. This includes a survey of a variety of fungi, which will introduce you
to various fungal structures, both vegetative and reproductive. You will later find that many of
these can act as opportunistic human and animal pathogens. All subsequent exercises will study
fungi and actinomycetes causing diseases in humans.
Equipment for each student or on each table
1. Microscope
2. Lens paper
3. Microscope slides
4. Microscope slide cover slips (glass)
5. Inoculating needle
6. Inoculating loop
7. Teasing needle
8. Forceps
9. 500 ml beaker with Quatsyl disinfectant—all used slides should be placed in here to
minimize possible release of pathogens
10. PVLG with aniline blue and other stains.
11. Slide box for your slide collection
2
General Laboratory Procedures
The following list of procedures is for the benefit of each student. Observance of these
minimal regulations will enhance your learning opportunity and reduce personal hazard.
Remember you are working with viable organisms, mostly pathogens.
1. Each student is responsible for the equipment provided. It will be checked back to the
instructor at the end of the semester.
2. All contaminated slides should be disposed of in a beaker containing disinfectant.
3. All contaminated glassware, tubes, and petri dishes will be disposed of in designated
containers. Do not discard any contaminated material in the trash can or waste basket.
4. If a culture is dropped and broken, do not attempt to clean the area; rather consult with the
laboratory instructor.
5. Avoid breathing in spores. Do not open plates to sniff to check for odors.
6. The laboratory working area for each student must be cleaned with Quatsyl disinfectant
before and after every laboratory period.
7. For laboratory assignments, students will be divided into groups of two or four, depending
upon the procedure to be completed. To maximize the learning process, it is necessary that
the workload be evenly distributed among the group members.
8. All culture transfers should be done in biological safety cabinets (hoods), unless specifically
allowed by the instructor
9. If you have any temporary or permanent problems with your immune system, please inform
the instructor privately.
Slide Collection
1. Should have one slide of each organism that we examine this semester, in the proper slot.
2. The slide collection will be graded on the following criteria:
a. Labels (6 pts.) – Label with labeling tape, the name neatly printed.
b. Sample Quality (6 pts.) – A good example of the fungus should be on the slide.
c. Slide Quality (6 pts.) – The slide should be properly and neatly prepared. PVLG
should be completely dry. Usually a few days at 60-70 C will dry the slide to
permanency.
d. Collection Content (6 pts.) – There should be a slide of each organism that we study.
***Slide boxes can be purchased for $10 (our cost) at the end of the semester, or you may take
the slides in some other box for your future reference.
3
Laboratory I - Survey of Fungal Structure
Objectives:
1. To learn techniques for the making of wet mounts.
2. To become familiar with fungal vegetative structures.
3. To become familiar with fungal reproductive structures.
Organisms: (1 agar plate for each 4 students)
Morchella
Schizophyllum commune
Mucor
Saccharomyces cerevisiae
Schizosaccharomyces octosporus
Aspergillus cristatus
Neurospora terricola
Aspergillus
Penicillium chrysogenum
Prepared slides of Rhizopus zygospores
Other prepared slides
You will be responsible for identifying all organisms in this lab to genus.
Equipment, Media and Reagents:
Slides & coverslips, Make permanent mounts using PVLG with aniline blue. Observe
these next week. For some we will only make temporary mounts with Aniline blue in
lactophenol
4
Procedures:
1. Prepare PVLG [with lactophenol aniline blue] preparations of each organism as directed by
the instructor and as below. These will be mostly tape mounts.
Morchella
Mucor
Saccharomyces cerevisiae
Schizosaccharomyces octosporus
Aspergillus cristatus
Neurospora terricola
drop coverslip on culture to observe hyphae in
situ
mount a tiny piece of the culture in PVLG
for sporangia:
For zygospores: tweeze out from center
interaction zone. Mount in PVLG
tape mount of culture, mount in PVLG
scrape, mount in PVLG
scrape, mount in PVLG
tape mount of culture, mount in PVLG
tape mount of culture, mount in PVLG
Aspergillus
Penicillium chrysogenum
tape mount of culture, mount in PVLG
tape mount of culture, mount in PVLG
Schizophyllum commune
Gilbertella
2. Examine each preparation for the structures indicated by the instructor. Remember that
“Mycology is an exercise in contemplative observation” (J. Rippon), so spend a lot of time
studying each culture and each slide you make. Make sure your drawings are large enough and
detailed enough to help your studying later. The more times you spend on this now the better
you will learn the fungi and the better you will do in the course.
3. Pure culture isolation. Take three Petri dishes containing PDA or SAB agar home with you.
Inoculate the Petri dishes with something interesting from somewhere you go in the next couple
days. After inoculation seal the petri dishes with parafilm and leave at room temperature in
filtered light (not direct sunlight), incubated agar side up. Bring the Petri dishes back to the next
lab. You will (eventually) be responsible for isolating three filamentous fungi in pure culture
and identifying them to species.
4. Observe books and other materials on demonstration. You should understand what the content
of each book is—i.e. what kinds of information you would expect to find there. Sometimes
knowing where to find the information is almost as important as knowing a piece of information.
5
Morchella
Gilbertella
Schizophyllum commune
Mucor
6
Saccharomyces cerevisiae
Aspergillus cristatus
Schizosaccharomyces octosporus
Neurospora terricola
7
Aspergillus fumigatus
Rhizopus sporangia prep slide
Penicillium chrysogenum
Rhizopus zygospores prep slide
8
Penicillium conidia on orange p.s.
Aspergillus conidia p.s.
Penicillium cleistothecium p.s.
Morchella asci p.s.
9
Coprinus basidia p.s.
Rhizopus spot plate
Aspergillus cristatus cleistothecia
10
Neurospora terricola perithecia.
Morchella asci whole mount.
11
12
13
Laboratory II - Common Fungal Contaminants
Objectives:
1. To learn how to prepare slide cultures.
2. To become familiar with and be able to identify some common fungal laboratory
contaminants.
Organisms: (1 slant culture for each 4 students)
Week 1 Absidia
Circinella
Mucor
Rhizopus stolonifer
Syncephalastrum
Aureobasidium pullulans
Geotrichum candidum
Rhodotorula gracilis
Cunninghamella
Week 2 –
Alternaria
Aspergillus niger
Cladosporium
Fusarium roseum
Drechslera
Paecilomyces
Penicillium chrysogenum
Scopulariopsis
Trichoderma
Acremonium
Curvularia
Verticillium
You will be responsible for identifying the organisms in these two labs to the taxonomic
level listed here
Equipment, Media, and Reagents:
Slides & coverslips
Sterile glass petri dishes for slide cultures
Sterile water
PVLG with aniline blue
Begin pure culture isolation as directed by your instructor. You will eventually be responsible for
identifying two filamentous unknowns.
14
Procedure:
1. Prepare a slide culture for each organism as described here and as demonstrated by
your instructor.
MYCOLOGY SLIDE CULTURE TECHNIQUE (See also Beneke and Rogers)
Principle
In the study of fungi, it is often necessary to observe the undisturbed relationship between
reproductive structures and the mycelium. This may be accomplished by growing the fungi on
glass slides in a moist chamber. You should constantly be aware of the possible infectious
hazards of this procedure. Consult the procedure described in Beneke and Rogers (1996)
1. Place a slide on a bent-glass rod in the bottom of a petri dish, add a cover slip and a piece of
brown paper or filter paper, cover and sterilize.
2. Prepare Sabouraud dextrose agar plates with about 10 ml of agar per plate. Allow to solidify
and dry. Cut agar blocks about 1 cm square or puncture with a test tube and lift.
3. Place the block of agar, using sterile technique, on the slide in a petri dish. Lift out with
flamed loop.
4. Inoculate the sides of the block with a small fragment of fungus being studied by cutting into
it.
5. . Sterilize coverslip by passing it briefly through a flame. Cover inoculated block with the
sterile cover slip.
6. Add sterile water to bottom of petri dish.
7. Incubate at 25 C until sporulation occurs (usually 1-2 weeks, occasionally faster). The slide
preparation may be checked periodically under the low power of a microscope.
8. When sporulation is complete, carefully lift off cover slip and place onto a drop of polyvinyl
aniline blue (PVLG) on a clean slide, making your first slide.
9. Cut away agar black from any hyphae that are coming from it. Lift agar block from slide and
discard.
10. Place a drop of PVLG in the center of growth on the slide and cover with a fresh cover slip.
11. Blot away excess mounting fluid from the two preparations, allow to dry, and place in oven
(60-70 C ) to dry for a minimum of 2 days. Usually drying for a week is best.
15
Absidia
Mucor
Circinella
Rhizopus stolonifer
16
Syncephalastrum
Geotrichum candidum
Aureobasidium pullulans
Rhodotorula gracilis
17
Alternaria
Aspergillus niger
Cladosporium
Fusarium roseum
18
Bipolaris or Drechslera
Penicillium chrysogenum
Paecilomyces
Scopulariopsis
19
Trichoderma
Acremonium
Curvularia
Verticillium
20
Laboratory III - Superficial Mycoses
The superficial mycoses include a few diseases caused by a heterogeneous group of organisms
that are usually saprophytes, but occasionally produce lesions on the skin or hair.
1. Piedra
A disease of the hair. Black Piedra is caused by Piedraia hortai and White Piedra is caused by
Trichosporon beigelii. The fungus infects the hair producing stony hard nodules along the hair
shaft. Observe demo slides.
2. Tinea versicolor (Pityriasis)
Causal organism - Malassezia furfur
A skin disease. The disease is characterized by fawn to brownish-colored lesions, usually arms,
neck, face, scalp, thighs, and groin area. No attempt is made to culture this lipophilic fungus in
the laboratory. Clinical diagnosis is confirmed by a microscopic examination of a potassium
hydroxide preparation of the scales from a lesion. Observe demo slide.
3. Tinea nigra palmaris
Causal organism - Exophiala werneckii
A superficial infection of the skin on the palm of the hand which is characterized by brown to
black macules resembling silver nitrate stains on the skin. The lesions are smooth, flat, without
raised borders, are not scaly or vesiculate. Brown hyphae will be seen in the stratum corneum
only.
Diagnosis: Scrape pigmented areas on palm and examine in a KOH preparation for brownish,
septate, branching hyphal segments.
In culture: Culture skin scrapings from lesion on Sabouraud’s glucose agar and incubate at
room temperature. On Sabouraud’s glucose agar the organism will develop into shiny, moist,
black yeastlike colonies with continued growth areas of grayish mycelium. Budding cells
develop from the slides of the hyphae, remain clustered and appear similar to clusters of
blastospores along the pseudohyphae of Candida sp.
4. Erythrasma
Causal organism - Corynebacterium minutissimum
A superficial infection with lesions primarily occurring in various intertriginous areas such as
perineal, crural, axillary, and submammary regions. Although the causal organism is a
bacterium, the infection is diagnosed by mycological methods - microscopic examination of skin
scrapings from the lesions. The organism appears short, delicate, branching filaments (less than
1 um in diameter) which are easily broken into shorter bacillary segments. Culturing of the
pathogen is not a routine laboratory procedure.
5. Trichomycosis axillaris
Causal organism - Corynebacterium tenuis
Another superficial dermatophytic infection caused by a bacterium. It is an infection of the
axillary and pubic hairs producing yellow, red, or black nodules which are usually scattered
21
along the hair shaft. Clinical diagnosis is by microscopic examination of potassium hydroxide
preparations of infected hair. The nodules on the hair consist of short, delicate, bacillary
elements. No attempt is made to culture the organism. Observe demo slide.
Objectives:
1. To become familiar with the superficial mycotic diseases listed above by observing
materials from books, displays and demo materials..
2. To become familiar with the cultural and microscopic characteristics of Trichosporon
beigelii and Exophiala werneckii.
Organisms: (1 slant culture for each 2 students)
Exophiala werneckii
Trichosporon beigelii
Cladosporium sp. ( a proteolytic saprophytic species)
Piedraia hortai prepared demo slide only
Equipment, Media and Reagents:
Slides & coverslips
PVLG with aniline blue
12% Gelatin tubes (2 for each 2 students)
12% Gelatin
Brain Heart Infusion Broth
Gelatin
Distilled water
Adjust pH 7.4 - 7.6
Tube - 6 ml in screw cap tubes
Sterilize, 15 min., 15 p.s.i.
25 g
120 g
1000 g
Demonstration materials for Black Piedra, Erythrasma, and Trichomyces axillaris
Procedure:
1. Inoculate one 12% gelatin tube with a large fragment of Exophiala werneckii and one
tube with Cladosporium sp. (Saprophytic species of Cladosporium are proteolytic, while
pathogenic species e.g. E. werneckii and C. carronii do not breakdown protein.)
2. Examine and study demonstrations.
22
1. Exophiala werneckii
a. Colony characteristics -
b. Gelatin test
1. Trichosporon beigelii
a. Colony characteristics -
23
2. Black Piedra - on hair. demo slide
4. Pityriasis versicolor - slide demonstration
24
5. Erythrasma - slide demonstration
6. Trichomycosis axillaris - slide demonstration
25
Laboratory IV - Dermatophytes
The dermatophytes are pathogenic fungi that produce cutaneous infections of the skin, hair and
nails, and are the most common and widely distributed of the fungal diseases of humans. The
maladies are referred to as a ringworm or tinea and are incited by species of Epidermophyton,
Microsporum, and Trichophyton. These organisms rarely invade subcutaneous tissues or internal
organs. The isolation and identification of the fungus involved is essential, especially in cases
that have been difficult to treat. With the advent of azole compounds, Lamisil, and their
different efficacies in treating infections, it has become essential to identify the fungus to species.
Many insurance companies will not pay for prescriptions if the fungus is not identified.
Clinical isolation Plate out scrapings from lesions or fragments of infected hair on Sabouraud glucose agar
containing chloramphenicol and cycloheximide (can use Mycosel or Mycobiotic agar).
Laboratory identification –
Identification is based upon colony and microscopic characteristics. The three genera are
differentiated by the shape of the macroconidia. Species identification is based upon various
morphological characteristics. In addition, sometimes physiological characteristics are also used.
It is also helpful to know from where on the body the scrapings or hair came from.
Organisms: (One slant culture for each 4 students) Not all species may be available for making
slides. Demonstration slides are available for most of these.
Week 1 -Epidermophyton floccosum
Microsporum audouinii
Microsporum canis
Microsporum fulvum
Microsporum gypseum
Microsporum nanum
Microsporum vanbreuseghemii
Microsporum gallinae
Week 2 -Trichophyton ajelloi
Trichophyton verrucosum
Trichophyton megninii
Trichophyton mentagrophytes
Trichophyton rubrum
Trichophyton schoenleinii
Trichophyton terrestre
Trichophyton tonsurans
Trichophyton equinum
Trichophyton simii
26
You are responsible for identifying the fungi on the preceding page to genus. Learn the variation
within each genus by looking at as many species as possible.
Equipment, Media and Reagents:
Slides & coverslips
Sterile glass petri dishes for slide cultures
Sterile water
PVLG with aniline blue
Rice grain plates
Guinea pig hair (sterile)
Horse hair or human hair (sterile)
10% sterile yeast extract
Procedure:
1. Prepare a slide culture of each dermatophyte.
2.. Inoculate rice grain plates.
a. Place a few small fragments of Microsporum audouinii on one rice plate and M. canis or M.
distortum on the second rice plate. Take care not to transfer any agar with the inoculum.
Inoculate hair. Take a couple pieces of your hair and inoculate M. gypseum and one other
species in two slide culture setups.
Observe demo slides of Ectothrix and Endothrix invasion of hair.
Observe demo materials of infected nails and whatever else is available.
27
DERMATOMYCOSES
Diagnosis
A direct microscopic examination of hair, skin and nails suspected of being
infected with fungi offers the most important and best means of diagnosis. The material
is collected between two glass slides that have been previously wrapped in paper and
sterilized. To examine the skin or nail scrapings or small “stubs” of hair, place the
material in a drop of 10-40% KOH on the end of a glass slide, apply a cover glass and
heat gently over a low flame. If the material fails to clear sufficiently, add more KOH to
the edge of the cover glass and gently heat again.
Although a clue to the genus Trichophyton or Microsporum may be obtained from
the characteristic appearance of the infected hair, the species can be determined only after
the fungus has been isolated in culture. Since the three genera Trichophyton,
Microsporum, and Epidermophyton are identical in appearance in infected skin and nails,
this material must be collected and cultured to obtain both a generic and specific
identification of the invading fungus. The material is cut into small fragments and three
or four pieces placed short distances apart on each of two or three Sabouraud’s glucose
agar slants. Cultures are maintained at room temperature, and must be kept for two or
three weeks before they can be considered negative.
Many of the dermatophytes do not produce distinguishing spore forms or
consistent pigment on Sabouraud’s medium. Recent nutritional studies have shown that
deficiencies play an important part in the growth of these fungi and the morphology
which they display.
A. T. rubrum – the reddish to purple pigment in the agar distinguishes this species from
T. mentagrophytes. Two media may be used for pigment production: Potato dextrose
agar (Difco) and corn meal dextrose (2%) agar.
Microscopic morphology is more consistent on Heart infusion agar plus tryptose (Blood
agar base, Difco).
B. Christensen’s Urea Agar. T. mentagrophytes is positive immediately while T. rubrum
takes some days to become positive.
C. Further differentiation of T. mentagrophytes from T. rubrum is based on the ability of
T. mentagrophytes to perforate hair in culture while T. rubrum does not. Sterilized
human hair is placed in a petri dish with sterile distilled water containing yeastextract (0.1 ml of 10% yeast extract in 25 ml distilled water). Inoculate with culture
to be tested, incubate at 25C for two weeks and examine hair in lactophenol cotton
blue.
D. T. verrucosum (T. faviforme) – isolate best on blood agar base (Difco) plus thiamine
(10 ml/liter) with incubation at 37C. Sabouraud’s glucose thiamine agar may also be
used.
Microscopic morphology, spore production, best on Sabouraud’s glucose thiamine agar at
room temperature.
E. T. tonsurans – characteristic gross morphology on Sabouraud’s glucose agar.
Microscopic morphology best on Wort agar at 7-10 days and again at 20-24 days.
F. T. violaceum – slow growing, glabrous pigmented culture on Sabouraud’s glucose
agar characteristic of this species.
28
TABLE I Fungi That Invade Hair, Skin and/or Nails
Fungus
Hair
Skin
Nails
Epidermophyton floccosum
-
+
+
Microsporum spp.
+
+
-
Trichophyton spp.
+
+
+
Malassezia furfur
-
+
-
Exophiala werneckii
-
+
-
Candida spp.*
-
+
+
+ = invades
- = does not invade * = may cause lesions of scalp
TABLE II
Type of Hair Invasion by Some Fungi
Ectothrix invasion
Endothrix invasion
Microsporum audouinii
Trichophyton schoenleinii
M. canis
T. tonsurans
M. ferrugineum
T. violaceum
M. gypseum
T. rubrum
Trichophyton mentagrophytes
T. soudanense
T. verrucosum
T. yaoundei
T. megninii
T. gourvillii
T. equinum
29
Epidermophyton floccosum
Microsporum audouinii
Microsporum canis
Microsporum fulvum
30
Microsporum gypseum
Microsporum nanum
Microsporum vanbreusighemii
Microsporum gallinae
31
Week 2 –
Trichophyton ajelloi
Trichophyton concentricum
Trichophyton verrucosum
Trichophyton megninii
32
Trichophyton mentagrophytes
Trichophyton rubrum
Trichophyton schoenleinii
Trichophyton terrestre
33
Trichophyton tonsurans
Trichophyton equinum
Trichophyton violaceum
Trichophyton simii
34
Laboratory V - Subcutaneous Mycoses
The subcutaneous mycoses are caused by fungi that probably exist as saprophytes in nature and
infect the skin and subcutaneous tissue, rarely involving internal organs in the body. The three
diseases of this type are chromoblastomycosis, mycetoma, and sporotrichosis. Usually a person
acquires infection accidentally as a result of some traumatic implantation.
Objectives:
1. To become familiar with the subcutaneous mycoses.
2. To become familiar with the cultural and microscopic characteristics of the fungi
associated with each of the subcutaneous mycoses.
3. To become familiar with the diagnostic procedures for each of the associated diseases.
Organisms: (slant culture for each 4 students)
Week 1 -- Fungi associated with chromoblastomycosis
Fonsecaea pedrosoi
Fonsecaea compacta
Phialophora verrucosa
Phialophora gougerotii
Phialophora richardsiae
Exophiala spinifera
Week 2 --
Fungi associated with mycotic mycetoma
Scedosporium apiospermum – (anamorph Pseudallescheria boydii)
Exophiala jeanselmei
Fungus causing sporotrichosis
Sporothrix schenckii
You are responsible for the species listed
Equipment, Media, and Reagents:
Slides & coverslips
Sterile glass petri dishes for slide cultures
Sterile water
PVLG with aniline blue
Gelatin tubes
Starch agar plates
35
Procedure:
Week 1 -- For chromoblastomycotic fungi
1. Prepare slide cultures for each species and incubate at room temperature. Examine after
sufficient growth has occurred (may take 2 to 3 weeks as these fungi grow relatively slowly).
Week 2 -- For mycetoma fungi
2. Prepare slide cultures for each species and incubate at room temperature. Examine after
sufficient growth has occurred.
3. Inoculate a gelatin medium tube and a starch medium plate with each organism. Inoculate
plates at room temperature. Be sure to include control organisms.
4. After fungus growth occurs on starch plate, flood with iodine solution. A clear zone around
colony indicates an area of hydrolysis.
Interpretation -Scedosporium apiospermum
Exophiala jeanselmei
Proteolytic Activity
+
0
Media -Gelatin medium
Brain heart infusion broth
Gelatin
Distilled water
Adjust pH to 7.2 - 7.4
Dispense 5 ml per tube and sterilize
Amylolytic Activity
+
0
25g
120 g
1000 ml
36
Exophiala dermatitidis
Phialophora verrucosa
Phialophora gougerotii
Phialophora richardsiae
37
Exophiala spinifera
Scedosporium apiospermum
Exophiala jeanselmei
Sporothrix schenckii
38
Week 1 – YEASTS Note: certain species may not be available. Check board.
1. Prepare slides for slide box.
2. India Ink
Cryptococcus neoformans
3. Germ tube test - rabbit serum
Candida albicans
Candida tropicalis
4. Chlamydospore production (1 plate/pair)
Candida albicans
Candida tropicalis
5. Ascospore production (3 tubes/pair)
Hansenula anomala
Saccharomyces cerevisiae
Schizosaccharomyces octosporus
6. Rapid nitrate (45C) (2 swabs/pair)
Cryptococcus terreus
Cryptococcus neoformans
7. CAFC disks
Candida albicans
Cryptococcus neoformans
1. Add water to slide plate
2. Place 2 CAFC discs on slide
3. Moisten disc with sterile water
4. Inoculate discs with test organisms. One with Cryptococcus neoformans, one with
Candida albicans
5. Incubate at 37 C and examine each hour for the production of brown pigment.
8. Urease test
Cryptococcus terreus
Candida glabrata
9. Candida albicans screen
Candida albicans
Cryptococcus laurentii
10. ChromAgar Candida 1 plate for each 4 people
Candida albicans, Candida krusei, Candida tropicalis, Cryptococcus neoformans
39
Week 2 YEASTS - Use Unknown provided to you
1. Delman plate (CM-Tween 80)
2. Urease slant
3. Carbohydrate assimilation plates
4. API 20C Aux. system if available
5. NO3 assimilation - next week if necessary
6. Germ tube - next week if necessary
7. ChromAgar Candida 1 plate for each 4 people
8. Cryptococcus latex test
40
Laboratory VI - Pathogenic Yeasts
Mycologically, the yeasts encompass a relatively large group of fungi. In a broad sense, the term
yeast refers to those fungi that possess a predominantly unicellular thallus, which reproduce
asexually by budding or fission. If sexual reproduction occurs they typically produce ascospores
in a naked ascus. In growth, yeast colonies typically resemble bacterial colonies and not
mycelioid fungal colonies.
In the diagnostic medical microbiology laboratory, the isolation of yeasts from clinical material
is an everyday occurrence. However, their identification as well as their significance often poses
problems for the technologist and the physician. With the constant use of antibiotics, steroids,
antimitotic and immunosuppressive drugs, yeasts have become a major group of opportunistic
pathogens. Several species of yeasts, including Candida albicans and Cryptococcus neoformans,
are now recognized as etiologic agents of disseminated fungal disease. This is especially true in
patients whose resistance has been altered by an initial disease (such as AIDS), chemotherapy or
certain surgical procedures. Some Rhodotorula species have been isolated from a number of
patients receiving intravenous medication. Candida glabrata is frequently isolated from urine
specimens. Candida parapsilosis, C. guilliermondii and C. tropicalis are seen in increasing
instances as etiologic agents of endocarditis in drug addicts and in patients undergoing some
form of cardiovascular surgery. Cryptococcus albidus and Cryptococcus laurentii have been
shown to cause respiratory disease as well as skin lesions. The incidence of meningitis caused
by species other than Cryptococcus neoformans is on the increase. Many laboratories now are
demonstrating Cryptococcus neoformans as a frequent causative agent in pneumonitis.
Identification of yeasts isolated from blood cultures, cerebral spinal fluid, a closed lesion or a
surgical specimen should be made. Such yeasts are unlikely to be contaminants. Yeasts from
the upper respiratory tract, lower urinary tract or the intestinal tract may represent normal flora,
but, in some instances, may contribute significantly to a patient’s disease process. The yeasts
isolated from any of these sources should be reported so that the physician can interpret their
significance.
The number of species of yeasts that the diagnostic laboratory should find necessary to identify
is not large. Seven Candida species, two Torulopsis species (usually included in Candida), two
Trichosporon species and four species of Cryptococcus represent the majority of the yeasts
isolated. To successfully identify a yeast, a combination of several tests must be employed.
These tests are feasible for even the smallest of laboratories and include:
1. Checking yeast for purity
2. Colony characteristics
3. Cell size and morphology
4. Chlamydospore production
5. Germ tube test
6. Fermentation - assimilation studies
7. Nitrate assimilation test
8. Urease production
9. India Ink preparation or nigrosin
10. Ascospore production
41
Objectives:
1. To become familiar with the human diseases caused by the yeasts.
2. To become familiar with colony characteristics and morphology of various pathogenic
yeasts.
3. To become familiar with the laboratory diagnostic procedures in identifying the
various pathogenic yeasts.
Organisms: (1 slant culture for each 4 students)
Candida albicans
Candida guilliermondii
Candida krusei
Candida parapsilosis
Candida kefyr
Candida stellatoidea
Candida tropicalis
Cryptococcus albidus
Cryptococcus neoformans
Cryptococcus terreus
Hansenula anomala
Rhodotorula rubra
Saccharomyces cerevisiae
Candida glabrata
Trichosporon beigelii
You are responsible for learning the methods used to distinguish the species and the kinds
of characters that are used. We don’t expect you to memorize the physiological test results,
but you should be able to interpret them.
Equipment, Media and Reagents:
Slides & coverslips
PVLG with aniline blue
India ink or nigrosin
Sterile pipettes (1 ml x 5 ml), Disposable pipettes
Sterile petri dishes
Germ sera tubes (0.5 ml of pooled human or bovine serum in each tube)
Sterile 0.85% saline
Fermentation broth tubes (dextrose, galactose, lactose, sucrose, maltose, & raffinose)
Assimilation discs (dextrose, galactose, lactose, sucrose, maltose, raffinose, Trehalose)
Blank discs
Yeast nitrogen base, Yeast carbon base
2% agar deeps
Saturated KNO3 solution
Chlamydospore formation agar (corn meal - Tween - 80)
Urea agar slants
Malt extract agar slants
Carbolfuchsin stain
95% ethyl alcohol containing 1% HCl
Aqueous methylene blue, Media and supplies for special tests
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Procedure:
1. Method to check yeast for purity.
a. Emulsify a loop full of material from the yeast colony in 5 ml of sterile distilled water and
streak a blood agar plate and a Sabouraud dextrose agar plate with the suspension.
b. Incubate the blood agar plate at 37 C and the Sabouraud agar plate at room temperature. If
after 48 hours no bacteria appear on the blood agar plate, the culture may be considered free of
bacteria.
c. If bacteria are present, purify as follows:
1. To a tube containing 10 ml of Sabouraud dextrose broth, add 1 drop of 1N; to a second tube
of broth add 2 drops of 1N HCl, 3 drops to a third tube, and 4 drops to a fourth tube.
2. Add a drop of yeast suspension to each of the four tubes and incuate at
room temperature for 24 hrs.
3. Subculture from each of the broth tubes to blood agar plates and incubate at
37
C for 48 hrs. One or more of the plates should be free of bacteria; if not, further attempts to
purify the culture is unlikely to succeed.
d. The initial Sabouraud agar streak plate should be incubated for 72-96 hrs. If all colonies on
the plate are identical, the yeast may be considered pure. If there is more than one type of colony
on the plate, each different colony type should be isolated, checked for purity and identified.
2. India ink preparation - to check for capsules
a. Mix a loopful of India ink (Pelikan) or nigrosin and a loopful of sterile distilled water on a
clean slide.
b. Transfer a small amount of yeast to the drop, emulsify, add a cover slip and examine for
capsules. The capsule is a clear area around the cell. Cryptococcus and Rhodotorula both
produce capsules. Rhodoturola species have reddish pigmented colonies. Remember that C.
neoformans tends to lose its capsule the longer it has been in culture.
3. Germ tube test
a. Touch the yeast colony with the tip of a disposable capillary pipette and insert pipette into
0.5 ml of pooled human or bovine serum. (Do not use heat coagulated serum) (Serum and
pipettes need not be sterilized.)
b. Incubate with pipette in serum at 37 C for 2 hrs. and 45 min., then examine a drop of the
mixture for germ tube production.
Approximately 98% of Candida albicans strains will produce germ tubes. After 3 hrs.
incubation, C. stellatoidea will produce germ tubes, therefore incubation time is very critical. C.
albicans and C. stellatoidea can be differentiated by a sucrose assimilation test. For all germ
tube negative yeasts, additional tests for identification must be conducted.
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4. Chlamydospore production
A chlamydospore is a thick-walled, intercalary or terminal cell. It is a characteristic structure of
Candida albicans.
a. With an inoculating needle, make two streak inoculations the entire length of a
chlamydospore agar plate; use just enough pressure to scarify the agar -- do not cut the agar.
b. With a loop, streak back and forth across the scarified area.
c. Place a sterile cover glass over a portion of the streaks.
Note: A strain of Candida albicans should also be inoculated on the plate as a control.
d. Incubate the plate at room temperature for 12-24 hours.
e. Examine the plate by removing the cover and placing the plate on the microscope substage.
Get the edge of the coverslip in focus under low power. When present, chlamydospores will
usually be found underneath the cover glass near the edge.
5 Ascospore production
a. With a loop, inoculate a malt agar slant or a V8 juice agar slant with the yeast culture.
b. Incubate at room temperature.
c. With a wet preparation, examine culture every 48-72 hrs. for 2-3 weeks. Sometimes
ascospores may not appear for 6 weeks.
An ascospore stain can be used
2. Flood slide with carbolfuchsin and steam gently for 2-3 min.
3. Wash and destain with 95% ethyl alcohol containing 1% HCl for a few seconds.
4. Wash and counterstain with aqueous methylene blue for 1-2 min. Wash, dry, and
examine. Ascospores will be red and vegetative cells will be blue.
6. Urease test
a. With a loop, inoculate a Christensen’s urea agar slant.
b. Incubate at room temperature and examine daily for 3-4 days.
If urease is positive, the medium will turn cherry red. Cryptococcus and Rhodotorula species are
urease positive. Also, an occasional isolate of Candida krusei, Candida humicola, and some
Trichosporon species may produce urease.
7. Fermentation Tests
Fermentation tests are not needed routinely to identify Candida species, particularly if
the technologist is familiar with the characteristic morphology of each Candida
species when examined on a chlamydospore agar plate.
a. Prepare a cell suspension of the yeast in 0.85% saline to a turbidity of a McFarland No. 1
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standard. A suspension giving 70% transmittancy at 365 nm on a B&L Spectromic 20
spectrophotometer is also satisfactory.
b. Inoculate each fermentation tube with 0.3 ml of the prepared suspension. Tighten caps
securely.
c. Incubate at room temperature for 14 days. Positive fermentation is recorded for the
production of gas only.
8. Carbohydrate assimilation test
This test checks which sugars can be assimilated by different species
a. Prepare a suspension of the yeast to be tested equivalent to a McFarland Standard number 4.
b. Flood the surface of the agar in a plate containing yeast nitrogen base and brom cresol purple
with the yeast suspension. Use about 1 ml. Remove the excess inoculum with a pipette and
leave the lid ajar for 10-15 minutes to allow the surface to dry.
c. Place three sugar impregnated discs (different sugars) equidistant apart on the agar surface of
one plate and three different sugar impregnated discs on the other plate.
d. Incubate at 30 C for 24-48 hours and read for evidence of carbohydrate utilization. A color
change from purple to yellow around the carbohydrate disk is indicative of the utilization of the
substrate. Carbohydrate disks may be surrounded by growth without a color change and this also
indicates utilization of the substance.
9. Nitrate assimilation test—we will not do this test. It has largely been supplanted by the
rapid nitrate test.
a. Prepare a cell suspension as for the carbohydrate assimilation test. (The yeast cells should be
starved in a yeast carbon base broth).
b. Add 0.3 ml of yeast suspension and 1.5 ml of yeast carbon base to the bottom of a sterile petri
dish.
c. Add 13.5 ml of liquid 2% water agar, thoroughly mix, and allow to harden.
d. Soak a sterile disc in a saturated solution of KNO3 and place on the agar in the center of the
petri dish.
e. Incubate at room temperature for 72 hrs. Assimilation of nitrate is recorded as growth around
the disc.
A known positive (Cryptococcus albidus) and a known negative (Cryptococcus neoformans) are
run in conjunction with the tested yeast as controls.
** Note: Nitrate assimilation tests need be run only on yeasts that are urease positive with
negative fermentation after 24 hrs.
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Some Methods for Rapid Yeast Identification Note: we will not do all these tests
1. Rapid urease test
a. Reconstitute a vial of Difco R broth with 3 ml of sterile distilled water on the day that
it is to be used.
b. Dispense 4-5 drops into each well to be used in a microtiter plate.
c. Transfer a heavy inoculum of each yeast colony to a well containing urea broth.
(Isolation of the yeast will be necessary if the culture is contaminated with bacteria.)
Colonies to be tested should not be older than 7 days.
d. In a laboratory situation, include a positive and negative control such as Cryptococcus
neoformans and Candida albicans respectively. Also include one well with uninoculated
broth only.
e. Seal wells with scotch tape and incubate for 4 hours at 37 C.
f. If urea broth shows any indication of having a pink color, it is positive for urease
production.
g. It is not necessary to test Rhodotorula species for urease production.
2. Rapid nitrate assimilation test
a. Sweep the tip of the swab impregnated with nitrate reduction medium across two to
three colonies of the yeast on a plate.
b. Swirl the inoculated swab against the bottom of an empty test tube to imbed the yeast
cells within the swab.
c. Incubate the tube and swab for 10 minutes at 45 C.
d. Remove swab from tube and add two drops each of a-naphthylamine and sulfanilic
acid reagents to the tube, then replace swab.
e. A change in color to red is positive for nitrate assimilation. (A known nitrate positive
and negative Cryptococcus should be used as controls.)
Nitrate Reduction Medium -Potassium nitrate
5g
Sodium phosphate, monobasic
11.7 g
Sodium phosphate, dibasic
1.14 g
Zepharin chloride
200 mg
Distilled water
200 ml
Standard, medium sized tipped, cotton swabs are saturated in the solution. Swabs are frozen,
lyophilized, and autoclaved. An alternative is to dry the swabs by vacuum for 24 hours and
autoclave. Swabs are stored in containers.
Nitrate Reduction Test Reagents -- It may take overnight for reagents to go into solution.
Frequent agitation helps
Reagent A
Reagent B
Glacial acetic acid
50 ml
Glacial acetic acid
50 ml
Water
125 ml
Water
125 ml
Sulfanilic acid
1.4 g
Dimethyl alpha napthylamine 1 g
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3. Brown pigment production by Cryptococcus neoformans
Cryptococcus neoformans produces a brown pigment (melanin) when grown on certain
specific substrates. Recent studies have shown that 95-98% of C. neoformans isolates are
positive for pigment production. Also, an occasional isolate of Trichosporon cutaneum
and Cryptococcus luteolus produces the pigment. C. neoformans can produce the brown
pigment (melanin) from various substrates containing certain ortho- and para-diphenols
and ortho-triphenols. The production of melanin from o-diphenol is due to 3, 4
dihydroxyphenylalanine phenoloxidase, which is an enzyme present in the cell wall of C.
neoformans.
Melanin pigment production is a fairly reliable test but false positives and false
negatives do occur, therefore, pigment production should only be used in combination
with other tests to provide a definitive identification of C. neoformans.
a. Caffeic acid-ferric citrate (CAFC) impregnated disks
1. For each organism to be tested, moisten one control disk (1 cm) and one CAFC
disk with sterile distilled water (2-3 small drops). Do not over saturate disks.
Place disks in a petri dish.
2. Transfer a loopful of Cryptococcus neoformans cells from a Sab agar slant to
each of the disks. Repeat with additional disks if other yeast are to be tested.
3. Incubate within an enclosed container at 37 C for 2-3 hrs. and check for the
production of brown pigment. If no pigment is seen, then check after 6 hrs. and
24 hrs. No brown coloration after 24 hrs. is interpreted as a negative reaction.
To prepare CAFC disks (note: prepared disks may also be purchased)
Stock solutions Caffeic acid - 1 mg of caffeic acid per ml in 0.5 M
Sorensen buffer (pH 7.0)
Ferric citrate - 0.5 mg of ferric citrate per ml distilled water.
To each disk (1 cm) add -1 ml of caffeic acid stock solution
0.5 ml of ferric citrate stock solution
3.5 ml of Sorensen phosphate buffer
(Control disks are saturated with Sorensen buffer only.)
Allow disks to dry overnight in sterile petri dishes at 37 C and store in dark bottles at room
temperature for no more than 2 months.
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4. API 20C AUX clinical yeast system
Within recent years several companies have prepared yeast diagnostic kits for a rapid
identification of clinically important yeast. The API 20C clinical yeast system is one method,
and has proven to be highly accurate and also has a shelf life of 12 months. The API 20C AUX
is a ready to use microtube system which consists of 19 assimilation tests and when combined
with computer assisted interpretation, allows for the identification of a wide range of yeast
species isolated in a clinical laboratory. The API 20C system consists of microtubes containing
dehydrated substrates for the assimilation reactions. Each microtube is inoculated with a basal
medium yeast cell suspension, incubated at 30C and read after 24, 48, and 72 hours. See the
sheets provided with the kit and included here.
3. ChromAgar http://www.chromagar.com/
A medium for the detection and differentiation of Candida, allowing easy detection of colonies
from different species.
CHROMagar Candida - will not only allow the growth and detection of yeasts - like traditional
media (Sabouraud) but in addition, just by the color of the colony, will instantly allow to
differentiate various Candida species.
CHROMagar Candida helps to recognize the major population of Candida infecting the patient
as well as for the first time - it offers a panoramic view on a mixed population with ability to
recognize the presence of a minor population within a patient.
Candida albicans- green
Candida tropicalis - steel blue
Candida krusei - rose, fuzzy
Inoculate plates as directed by your instructor.
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Laboratory VII- Systemic Pathogenic Fungi
The systemic mycoses may involve all or any of the internal organs of the body. Skin,
subcutaneous tissue, and bone may also be involved. Disease symptoms vary from mild to quite
severe. Death may also occur if the patient does not respond to therapies.
The fungi involved in systemic mycoses are dimorphic organisms. In tissue and usually when
cultured at 37C they exhibit one phase (usually a yeast) and when cultured at room temperature
they produce a different phase. (mycelial). Fungi causing histoplasmosis, blastomycosis,
coccidioidomycosis and paracoccidioidomycosis are typical examples of this phenomenon.
Objectives
1. To become familiar with the systemic mycoses
2. To become familiar with the morphological characteristics of the true pathogenic fungi
3. To become aware of the hazards of working with the true pathogenic fungi
4. To become familiar with the proper laboratory techniques in handling the systemic fungi.
Organisms:
Histoplasma capsulatum
Blastomyces dermatitidis
Coccidioides immitis
Paracoccidioides brasiliensis
Sepedonium chryospermum
You will be responsible for identifying all the organisms here to species
Equipment
Slides +coverslips
Multitude of demonstration slides.
These fungi are too pathogenic for us to work with with the equipment we have available.
However, we are lucky to have many demonstration slides of these organisms, both mounts of
the cultures and in histopathological section. Observe all materials available.
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Histoplasma capsulatum
rt
Blastomyces dermatitidis rt
Histoplasma capsulatum 37C
Blastomyces dermatitidis 37C
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Coccidioides immitis rt
Coccidioides immitis 37C
Paracoccidioides brasiliensis rt Paracoccidioides brasiliensis 37C
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Aspergillus
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