Clinical Laboratory Science Program
CLS419 – Clinical Microbiology II
Mycology
Rotation II Manual
2011-2012
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Table of Contents
Section One
Schedule
3-4
Section Two
Basics of Mycology
5-21
Yeast
22-31
Section Four
Opportunistic Molds
32-40
Section Five
Dermatophytes
41-49
Subcutaneous Fungi
50-59
Systemic Dimorphic Fungi
60-67
Section Eight
Appendix
Mycology Procedures
Mycology Lab Worksheet
Univ. of WA Mycology Tutor, Table of Contents
68
69-70
71-72
73-74
Section Three
Section Six
Section Seven
CLS419 Clinical Microbiology II
Rotation II
Mycology
Table of Contents
Page 1
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section One:
Schedule
CLS419 Clinical Microbiology II
Rotation II
Mycology
Table of Contents
Page 2
Mycology Schedule
Reading Assignments:
 Textbook of Diagnostic Microbiology, Mahon & Manuselis, 4th edition, Chapter 27, pages 603 –
638.
 Your clinical site will also have a variety of valuable resources such as other textbooks, Mycology
atlases (great pictures of fungi), and clinical expertise that you may utilize during this week
Websites:
 University of Washington's Lab Training – Mycology (www.medtraining.org/ltl/)
Monday
Basics of Mycology Unit
 Watch lecture on Blackboard
 Read procedures provided in Manual and on Blackboard
 Do Study Questions
 Watch videos on Blackboard
o Safety (3:00 minutes)
o Scotch Tape Preparation (4:15 minutes)
o Tease Preparation (5:00 minutes)
o Slide Culture Setup (9:45 minutes)
o Slide Culture Take-down (3:00 minutes)
 Website: Lab Training - Mycology (Univ. of Washington) - Read the following sections
(check off as you go from this Table of Contents - 2 pdf pages):
o Introduction
o Fungal Detection
o Organism Identification
Yeast Unit
 Watch lecture on Blackboard
 Do Case Studies & Study Questions
 Website: Lab Training - Mycology (Univ. of Washington) - Read the following sections:
o Opportunistic Fungi - Overview & Candida albicans
o Disease Associations - Superficial Mycoses - Tinea versicolor
o Disease Associations - Systemic Mycoses - Cryptococcosis
 Lab: (Record all results on "Mycology Lab Worksheet" - please document all forms seen)
 Look at gram stain slides of yeast that are available in your slide box:
o Candida albicans
o Cryptococcus neoformans
Tuesday
Opportunistic Molds Unit
 Watch lecture on Blackboard
 Do Case Studies & Study Questions
 Website: Lab Training - Mycology (Univ. of Washington) - Read the following sections:
o Opportunistic Fungi - Aspergillus fumigatus, Aspergillus flavus, Rhizopus, Mucor,
Alternaria, Fusarium, Penicillium
 Lab: Look at LPCB preps of opportunistic molds (Record all results on "Mycology Lab
Worksheet" - please document all forms seen) that are available in your slide box:
o Rhizopus sp.
o Aspergillus fumigatus
o Aspergillus niger
o Penicillium sp.
o Fusarium sp.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Schedule
Page 3
Dermatophytes Unit
 Watch lecture on Blackboard
 Do Case Studies & Study Questions
 Website: Lab Training - Mycology (Univ. of Washington) - Read the following sections:
o Disease Associations - Superficial Mycoses - Dematophytosis
 Lab: Look at LPCB preps of dermatophytes (Record all results on "Mycology Lab
Worksheet" - please document all forms seen) that are available in your slide box:
o Microsporum canis
o Microsporum gypseum
o Trichophyton sp.
Review previous day’s material
Wednesday
Subcutaneous Fungi Unit
 Watch lecture on Blackboard
 Do Case Studies & Study Questions
 Website: Lab Training - Mycology (Univ. of Washington) - Read the following sections:
o Disease Associations - Subcutaneous Mycoses (Sporotrichosis,
Chromoblastomycosis, Mycetomas)
o Opportunistic Fungi - Acremonium species
 Lab: Look at slides of subcutaneous fungi (Record all results on "Mycology Lab
Worksheet" - document all forms seen) that are available in your slide box:
o Cladosporium, Acrotheca &/or Phialophora forms - LPCB prep
o Nocardia sp. - Gram stain
o Nocardia sp. - acid fast stain
Systemic Dimorphic Fungi Unit
 Watch lecture on Blackboard
 Do Case Studies & Study Questions
 Website: Lab Training - Mycology (Univ. of Washington) - Read the following sections:
 Disease Associations - Systemic Mycoses - Blastomycosis, Coccidioidomycosis,
Histoplasmosis
 Lab: Look at slides of systemic dimorphic fungi (Record all results on "Mycology Lab
Worksheet" - document all forms seen) that are available in your slide box:
o Histoplasma capsulatum - mold (LPCB prep)
o Histoplasma capsulatum - yeast (GMS stain)
o Blastomyces dermatitidis - yeast (LPCB prep)
Review previous days’ material
Thursday
Review:
 All objectives, case studies, and study questions
 All learning materials including websites and textbook
 Mycology Review PowerPoint on Blackboard – recommended that you use this as a
“mock practical”
Lab:
 Review all yeast/mold slides
Friday
Review:
 All learning materials including websites and textbook
Take Mycology Exam and Practical
CLS419 Clinical Microbiology II
Rotation II
Mycology
Schedule
Page 4
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Two:
Basics of Mycology
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 5
BASICS OF MYCOLOGY
Objectives:
Upon completion of this CLS 419 unit, the Clinical Laboratory Science student will:
1.
Discuss the following requirements of fungi:
a. Nutrients
b. pH
c. Temperature
d. Oxygen
2.
Compare the two growth forms of fungi:
a. Yeast
b. Mold
3.
Define the following terms:
a. Eukaryote
b. Prokaryote
c. Perfect fungi
d. Imperfect fungi
e. Hyphae
1) Septate
2) Aseptate (sparsely septate)
f.
Mycelium
1) Hyaline
2) Dematiaceous
g. Dimorphism
4.
Identify the following non-reproductive vegetative hyphal structures:
a. Favic chandeliers
b. Nodular organs
c. Racquet hyphae
d. Spiral hyphae
e. Rhizoids
5.
Identify the following asexual reproductive structures:
a. Blastoconidia
b. Conidia
c. Chlamydoconidia
d. Arthroconidia
e. Sporangium
6.
Describe appropriate specimen collection and processing requirements, including appropriate
media and incubation for the following:
a. Respiratory specimens
b. Urine
c. Hair, nail and skin scrapings
d. Tissue biopsy
e. Body fluids
f.
Exudates (wounds)
7.
Interpret the following stains and direct preparations, including the proper uses for each:
a. Lactophenol aniline blue
1) Tease mount
2) Scotch tape prep
3)Slide culture
b. Potassium hydroxide prep (KOH)
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 6
c.
d.
e.
Methenamine silver stain (GMS)
Periodic acid-Schiff stain (PAS)
Modified acid fast stain
8.
Evaluate the purpose, proper use, inhibitory or selective properties, significant ingredients and
colonial appearance of the following media:
a. Sabouraud dextrose agar (Emmon’s)
b. Mycosel agar
c. Inhibitory mold agar (IMA)
d. Niger seed agar (TOC) or birdseed agar
e. Brain heart infusion with blood agar (BHI)
f.
Potato dextrose agar
g. Cornmeal-Tween 80 agar (CMT)
9.
a.
Discuss the various approaches used in the laboratory identification of fungal cultures, including:
Length of time for growth
b. Growth characteristics (media, temperature, etc.)
c. Macroscopic appearance
d. Microscopic appearance
10. Discuss the appropriateness of anti-fungal susceptibility testing.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 7
Basics of Mycology
I.
Introduction
A. Mycology is the study of fungi
B. Mycoses are fungal diseases
1. Superficial & Cutaneous mycoses
a. Involves only hair, skin and nails
b. Little or no pathology; main worry is cosmetic effect
c. Involves destruction of the keratin of hair, skin and nails
d. Primarily caused by the Dermatophytes
2. Subcutaneous mycoses
a. Involves skin, muscle and connective tissue immediately below the skin
3. Systemic (deep-seated) / disseminated mycoses
a. Caused by pathogenic fungi that are highly virulent
b. Involves the deep tissues and internal organs, and has the ability to spread widely
throughout the body
c. Frequently initial site of infection is the lung
4. Opportunistic mycoses
a. Caused by ubiquitous saprophytic (“non-pathogenic”) fungi and occasionally
pathogenic fungi, all of which have limited virulence
b. Usually see in immunocompromised or debilitated patients
c. Causes subcutaneous and disseminated infections
C. Patients at risk for fungal infections
1. Immunosuppressed individuals are at highest risk (i.e. AIDS, decreased PMNs)
2. Organ transplant patients and others with previous treatment with corticosteroids,
cytotoxic agents, or prolonged antibiotic therapy
3. Patients with malignant neoplasms
4. Patients with various debilitating immunologic and metabolic disorders (i.e. SLE,
diabetes)
5. Occupations & activities involving direct skin contact with infected animals/materials and
ingestion or inhalation of contaminated aerosols/dust
D. Natural habitat is soil and vegetation
E. Taxonomy / Classification
1. By disease
2. By class
II. Characteristics of Fungi
A. Eukaryotic
1. Has a nucleus, nuclear membrane, endoplasmic reticulum, Golgi apparatus, and
mitochondria
2. Rigid cell wall containing chitin, mannans, and sometimes cellulose
3. Lacks chlorophyll
B. Growth Requirements
1. Nutrients – must absorb from environment since lack chlorophyll
2. pH – prefer neutral but tolerate wide range
3. Temperature – optimal growth at room temperature to 30°C, 37°C for dimorphic yeast
4. Oxygen – most are obligate aerobes
5. Moisture needed to grow, able to survive dry conditions with spores/conidia
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 8
C. Forms
1. Mould / Mold
a. Colony – growth of hyphae which form a matt of growth called the mycelium
b. Cells – multiple cells forming a filamentous mycelium
c. Reproduce either asexually (vegetative sporulation or aerial sporulation) or sexually
(sexual sporulation)
2. Yeast
a. Colony – bacteria-like, moist, smooth, creamy colonies
b. Cells – single, round to oval cells
c. Reproduce asexually by budding to form blastoconidia
 Pseudohyphae – elongation of blastoconidia showing sausage-like constrictions
between segments (true hyphae are not constricted at ends)
3. Dimorphism
a. Fungi that have the ability to exist in two forms depending on growth conditions
b. Generally dimorphic fungi have a mould phase and either a yeast or spherule phase
 Yeast /tissue phase – grows best at 37°C
 Mould phase – grows best at room temperature or 30°C
D. Structures
1. Hyphae
a. Long strand of tube-like structures
b. Types
 Aseptate (or sparsely septate) –
without (or very few) transverse walls
c.
Pigmentation
 Hyaline – Light colored hyphae
and/or conidia (fungi with septate
hyphae) due to no pigmentation or
brightly pigmented

Septate – subdivided into individual
cells by transverse walls

Dematiaceous – Dark colored
(brown-black) hyphae and conidia
(fungi with septate hyphae) due to
presence of melanin in cell wall
2. Mycelium
a. Mass of branching intertwined hyphae forming a matt of growth
b. Types
 Aerial mycelium (also called
reproductive mycelium)
o Portion of mycelium that projects
above the agar surface
o Special spore or conidiabearing fruiting bodies derive
from this portion
 Vegetative mycelium
o Extends into substratum of agar
and is responsible for absorbing
water and nutrients
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 9
o
Structures
Rhizoids – root-like structures
that may be located at the base of
a sporangiophore or internodally
along the hyphae.
Favic chandeliers – resemble
antlers of a deer, ends are blunt
and branched
Nodular organs – knots of
twisted hyphae
Racquet hyphae – resemble
tennis racquets with smaller end
attached to large end of an
adjacent club-shaped hyphae
Spiral hyphae – coiled or
corkscrew-like turns in hyphae
3. Vegetative reproductive structures
 Arthroconidia (arthrospores)
o Thick-walled barrel-shaped conidia produced by
fragmentation of the hyphal strand through the septation
points. They may form adjacent to each other or may be
separated by alternating empty spaces.

Blastoconidia (daughter cells)
o Budding forms characteristically produced by yeast
o A bud scar (dysjunctor) often remains at point where
conidium detached

Chlamydoconidia (Chlamydospores)
o Formed from pre-existing cells in the hyphae, which
become thickened and enlarged
o May be found within (intercalary), along the side
(sessile), or at the tip (terminal)
4. Aerial reproductive structures (fruiting bodies)
 Sporangiospores
o Spores contained in a closed sac called a sporangium
o The sporangium is supported on a base, termed the
columella, which is located at the tip of the specialized
hyphal segment called the sporangiophore
o This type of sporulation is characteristic of the
Zygomycetes

Conidia
o Spores produced on the surface of an elaborate fruiting body supported by a
specialized hyphal segment called a conidiophore
o The conidiophore can branch into secondary segments called metulae which
can become conidia-producing segments called phialides
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 10

Phialoconidia
o Conidia which arise from a tube or vase-shaped
structure called a phialide
o This type of sporulation is characteristic of the
Penicillium species

Annelloconidia
o Conidia which arise from a tube or vase-shaped
structure termed a annellide
o The tip of the phialide cyclically extends and
retracts when conidia form, leaving a succession of
scars
o Conidia may be formed singly, in long chains, or in
tightly bound clusters

Macroconidia
o Larger, multi-celled conidia that can vary in size and
shape
o The term “macro” should only be used when smaller
conidia are present.
Microconidia
o Tiny one-celled conidia, usually borne either directly from
side of hyphae or supported by a hair-like conidiophore
o The term “micro” is used only when larger conidia are
present

5. Sexual reproductive structures
a. Fungi having a sexual stage are termed “Perfect Fungi”
b. Fungi lacking a sexual stage are termed “Fungi Imperfecti”
c. Sexual sporulation requires 2 specialized fertile cells (having undergone meiosis) to
merge and have nuclear recombination occur on the
aerial hyphae
 Ascospores
o Sexual spores (meiotic division) produced in a
sac-like structure called an ascus. The ascus is
the sexual mother cell that forms ascospores
inside and may be protected on the outside by an
cleistothecium. The cleistothecium is a
protective sac within which asci and ascospores
form.
 Oospores
o Fusion of 2 morphologically identical cells from
different hyphal segments
 Zygospores
o Fusion of 2 morphologically identical cells from
the same hyphal structure
o Zygomycetes reproduce sexually in this manner
III. Specimen Collection & Transport
A. Specimen types and collection
1. Blood and bone marrow
a. Acquire by sterile technique
b. Inoculate biphasic agar-broth bottles designed specifically for fungal cultures
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 11
2. CSF
a. Acquire by sterile technique and transport in sterile container
b. Centrifuge and use sediment to make slides and inoculate media
c. Keep at room temperature is culture setup is delayed
3. Cutaneous: Hair, nail and skin scrapings
a. Hair
 Use Wood’s lamp to see infected areas. Pull out hair by roots with sterile
tweezers.
 If no fluorescence, pull out hairs that are broken and scaly
 Transport in sterile container
b. Nails
 Clean with 70% alcohol
 Scrape away and dispose of outer layers of nail
 Sample from beneath the nail plate to obtain softened material from nail bed, or
collect shavings from deeper portions
 Place into sterile container
c. Skin
 Clean with 70% alcohol
 If lesion present, scrape the actively growing edge
 Scrape areas that look most infected
4. Respiratory: bronchial washings, sputum, throat, transtracheal aspirates
a. Early morning specimen is best
b. 24 hour collections unacceptable (bacterial overgrowth)
c. Transtracheal aspirate should eliminate throat flora
d. Prepare slides for stains
5. Tissue biopsies
a. Collected by physician and should be kept moist with sterile saline in a sterile
container until processed
b. Should include normal tissue and tissue from the center and edge of lesion
c. Inspect tissue for granules and areas of pus and necrosis
d. Mince tissues for inoculation to media especially if Zygomycetes are suspected
6. Urine
a. Early morning clean catch or catheterized specimen is best
b. 24 hour collections unacceptable (bacterial overgrowth)
c. Centrifuge specimen and inoculate media with sediment
7. Vaginal, uterine cervix, prostatic secretions
a. Acquire by sterile technique and transport in sterile container
8. Wounds, subcutaneous lesions, mucocutaneous lesions, exudates
a. Acquire by sterile technique and transport in sterile container
b. From cysts and abscesses, material should be aspirated if possible
c. Examine for granules
B. Specimen Collection Issues
1. Collect from area most likely to be affected
2. Use sterile technique, avoid contamination with hands
3. Specimen must be adequate, reduces contamination
4. Keep specimen moist
5. Specimen must be properly labeled
6. Exact source/site aids in identification
7. Specimen must be delivered promptly to lab and processed quickly
a. Prevents overgrowth of bacteria and ubiquitous molds
b. Pathogenic molds can be slow growers
c. Yeast multiply quickly so refrigerate if delay in setting up culture
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 12
IV. Direct Examination of Specimens
 Provides rapid preliminary report and immediate presumptive diagnosis to guide the
physician in treatment
 Special morphological characteristics may give clues to the identity of the causative agent
 Aids in selection of special media to inoculate specimen to
 Direct exam may show evidence of infection despite negative cultures
 Allows for observation of yeast phase of dimorphic organisms
 May indicate need for more than one type of direct examination to be performed
A. Saline wet mount
1. Phase-contrast microscope is valuable or can use low light
2. Look for fungal elements such as:
a. Budding yeast with pseudohyphae (Candida)
b. Broad base budding yeast (Blastomyces dermatitidis)
c. Spherules (Coccidioides immitis)
d. Capsules (Cryptococcus neoformans)
B. Lactophenol aniline blue (LPAB) wet mount
1. Phenol kills organisms
2. Lactic acid preserves fungal structures
3. Aniline blue stains the chitin in fungal cell walls
4. LPAB prep can be made permanent
5. Look for fungal elements
C. Potassium hydroxide (KOH) preparation (10%)
1. Hair, skin or nail specimens
 KOH dissolves the keratin to make fungi more visible
2. Specimens containing cellular material such as sputum or vaginal secretions
 KOH dissolves the cells in background to make yeast / fungal elements more visible
3. Procedure
a. Add a drop of 10% KOH to specimen on slide. Coverslip.
 Gentle heating may aid in dissolving debris
 If specimen is thick, it may take 15-30 minutes to dissolve
b. Observe under low light or with phase-contrast microscope
D. Gram stain
1. Fungi stain gram positive
2. Look for yeast and fungal elements such as pseudohyphae
3. True fungi are 2-3 times wider than GPR’s and will not stain solidly inside
4. Capsule around yeast can prevent the definitive staining of the yeast itself
E. Acid-fast stain
1. Nocardia is partially positive with a modified Kinyoun acid-fast stain
2. Ascospores of Saccharomyces cerevisiae are acid-fast positive
F. India ink preparation
1. Used to observe for capsules around yeast (esp. Cryptococcus neoformans)
2. Procedure
a. Mix small drop of India ink with a drop of specimen and coverslip. (Strive for a thin
smear)
b. Let sit (up to 10 minutes) to allow cells to settle
c. Observe under microscope with condenser adjusted for maximum light. Look for a
clear capsule around yeast. Background is dark.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 13
G. Calcofluor white stain
1. Binds to polysaccharides in fungal cell walls
2. Fluoresces when exposed to UV light
3. 10% KOH can be added to dissolve background
4. Procedure
a. Add drop of Calcofluor White stain to specimen on slide. Coverslip.
b. Allow to sit approximately 3 minutes.
c. Use a fluorescent microscope and look for apple green fluorescence.
H. Tissue / Histological stains
1. Wright’s stain – look for intracellular yeast in tissue and bone marrow (Histoplasma
capsulatum)
2. Gomori Methenamine Silver (GMS) stain – fungi, Pneumocystis, and Actinomyces stain
black against a green background
3. Periodic Acid Schiff (PAS) stain – fungal elements are magenta against a light pink or
green background
V. Selection and Inoculation of Culture Media
A. Safety
1. Tube media preferred over plate media
a. Tube media will not dry out over long incubation periods
b. Reduces chance for fungal reproductive structure to become airborne and
contaminate the room and people
c. Never use plates when suspect Coccidioides immitis (extremely infectious and
aerosols may be inhaled)
2. ALWAYS work under a biological safety cabinet
3. Wear gloves and lab coat
4. Autoclave specimens and inoculated media when finished
5. Disinfect work area daily
B. Primary isolation media for fungi
1. Goal is to isolate all possible pathogens
2. Generally want 2 types of media – a nonselective media and a selective media (with
antibiotics to inhibit growth of bacteria and enriched for more fastidious fungi)
3. Ingredients required for fungal growth include carbon, nitrogen, vitamins, minerals and
amino acids
4. Nonselective Media
a. Brain heart infusion (BHI) with/without 5% blood
 Primary recovery of saprophytic and dimorphic fungi
 Useful for isolation of Histoplasma and Nocardia (media containing blood)
 Useful to convert dimorphic molds from mold to yeast phase when incubated at
35ºC
 Antibiotics (cycloheximide & chloramphenicol) can be added to make media
selective for dimorphic moulds
b. Inhibitory mold agar (IMA)
 The best medium to isolate fungal opportunists from a non-sterile site
 Primary recovery of dimorphic pathogenic fungi and saprophytic fungi that are
inhibited by cycloheximide
 Chloramphenicol & gentamicin inhibit growth of bacteria
c.
Sabouraud’s brain heart infusion agar (SABHI)
 Primary recovery of saprophytic and dimorphic pathogenic fungi, particularly
fastidious strains
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 14
5. Selective Media
a. Mycosel agar
 Selective for isolation of dermatophytes
 Chloramphenicol inhibits bacteria and Nocardia
 Cycloheximide inhibits rapid saprophytes and:
o Aspergillus fumigatus (25-60%)
o Cryptococcus neoformans
o Candida krusei
o Pseudallescheria boydii
o Candida tropicalis
o Nocardia asteroids
o Candida parapsilosis
o Piedraia hortae
o Trichosporon beigelii (cutaneous)
b. Dermatophyte test medium (DTM)
 Screening media for dermatophytes
 pH change causes the phenol red indicator to change from yellow to red
 Contains antibiotics
6. Incubation temperature
a. 30ºC is best (room temperature = 25ºC, is acceptable, some fungi may multiply
slower at this temperature)
b. 37ºC may inhibit some fungi, but necessary for yeast phase of dimorphic fungi
7. Incubation time
a. Hold cultures for 4-6 weeks, examining twice weekly for growth
b. Dependent on media, temperature and inhibitors in the specimen
8. Incubation atmosphere
a. Moist – 40-50% relative humidity
b. Ambient air
C. Subculture and special identification media for fungi
1. Once fungi have grown on primary culture, one frequently needs to subculture for
complete isolation and identification
2. Media
a. Sabouraud dextrose agar (SDA)
 Supports growth of all fungi (except Histoplasma and Nocardia)
 Consists of dextrose, peptone, agar and water
 pH 5.6 to inhibit bacteria which prefer pH 7.2
b. Neutral Sabouraud dextrose agar (Emmon’s modification)
 Subculture yeast, allows for better maintenance of yeasts
 Less dextrose and a neutral pH compared to regular SDA
c. Cornmeal-Tween 80 agar (CMT)
 Promotes hyphal and blastoconidia formation
 Observe pseudohyphae & chlamydoconidia production by Candida albicans
 Enhances pigment of Trichophyton rubrum when 1% glucose is added
 Procedure
o With loop, make one streak into the agar down the
center of an area and 3 or 4 parallel cuts across the
first ½ inch or 1 cm apart, holding the inoculating
wire at about a 45º angle to dilute inoculum.
o Incubate 24-72 hours at 30ºC
o After incubation, place coverslip on surface of the
agar, covering inoculation streaks
o Examine growth through the coverslip with the
microscope using the 10x and 40x objectives. Look for the most
characteristic morphology near the outer edges of the coverslip.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 15
d. Niger seed agar or Birdseed agar
 Used for isolation of Cryptococcus neoformans from contaminated specimens
 Cryptococcus neoformans produces phenoloxidase enzymes. These enzymes
break down the substrate caffeic acid forming a brown pigment
e. Tween 80 / Oxgall / caffeic acid agar (TOC)
 Observe brown pigment production by Cryptococcus neoformans
 Can observe germ tube production by Candida albicans
 Better chlamydoconidia development than Cornmeal/Tween 80
f. Potato dextrose agar
 Stimulates spore formation and pigmentation
 Used to subculture fungi for slide culture and observe for colony morphology
VI. Examining the Fungal Culture
A. Differentiating Pathogenic fungi
1. Growth rate is 10 days or more (slow growers)
2. Growth on Mycosel agar
3. Color: dull buff, brown, mousey gray
4. Dimorphic
a. Mold phase grows at 30°C (room temperature)
b. Yeast phase grows at 35°C on BHI agar
B. Identification of fungi
1. Growth rate
a. Rapid = 1-5 days
b. Intermediate = 6-10 days
c. Slow = 11-28 days
2. Colonial morphologic features
a. Appearance (topography)
 Rugose – colonies have deep furrows irregularly
radiating from the center
 Umbonate – colonies have a button-like central
elevation
 Verrucose – colonies have a wrinkled, convoluted
surface
 Flat
b. Texture
 Cottony (wooly) – very high, dense aerial
mycelium
 Glabrous (waxy) – smooth surface due to no
aerial mycelium (yeast-like)
 Granular (powdery) – flat and crumbly due to
dense conidia production
 Velvety – colonies produce low aerial mycelium
c. Pigmentation
 Observe color on both surface of colony and on
reverse side of plate
3. Microscopic morphologic features
a. Most definitive means of identification
b. Evaluate:
 Shape
 Method of production
 Arrangement of conidia/spores
 Size and color of hyphae
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 16
4. Microscopic techniques for evaluating fungi
a. Tease mount
 Procedure
o Using two sterile teasing needles, transfer a portion of colony (middle third)
to a slide
o Gently tease mycelium apart with teasing needles
o Add a drop of Lactophenol aniline blue stain
o Coverslip and observe for fruiting structures under light microscope at 10x
and 40x
 Advantage
o Perform and examine immediately after maturation
 Disadvantage
o Structural morphology is disturbed
b. Scotch tape preparation
 Procedure
o Lightly touch transparent scotch tape, sticky side down, to surface of colony
and then removing it
o Place a drop of Lactophenol aniline blue stain onto a slide
o Affix tape, sticky side down, into the stain on the slide
o Observe for fruiting structures under light microscope at 10x and 40x
 Advantages
o Perform and examine immediately after maturation
o Retains juxtaposition of spores and hyphal elements
 Disadvantages
o Prep is not easily preserved (view within 30 minutes and then discard slide)
o Contamination can occur
c.
Slide culture
 Procedure
o Place glass slide on 2 wooden sticks in Petri dish (gauze or paper towel
under sticks moistened with sterile water)
o Using sterile scalpel, cut 1 cm x 1 cm square of SAB or Potato dextrose agar
and place on slide. Two pieces of agar can be placed on the slide to provide
duplicate cultures.
o Inoculate the 4 sides of the agar with mould using teasing needles or sterile
wooden stick
o Place coverslip on top of agar
o Tape plate shut and incubate at room temperature (22°C)
o Examine for growth periodically & add more water as needed to keep moist
o When conidia / spores are evident, carefully lift coverslip off agar using
forceps and place onto slide containing a drop of lactophenol aniline blue
stain (coverslip can be sealed with fingernail polish to keep slide
permanently)
o Observe under light microscope at 10x and 40x
 Advantages
o Fungal elements are grown and maintained in their original juxtaposition,
making identification easier
o Two mounts from one culture, so you can view one slide and if necessary,
leave the other slide to incubate longer

Disadvantages
o Technical expertise required
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 17
o
o
VII.
Must wait for fungus to mature on inoculated media before identification can
occur
Zygomycetes grow past coverslip before forming reproductive structures
Serologic Diagnosis of Fungal Disease
 Generally performed only in select reference laboratories
A. Immunodiffusion
1. Aspergillus
2. Blastomyces
3. Histoplasmosis
B. Complement fixation
1. Blastomyces
2. Coccidioidomycosis
3. Histoplasmosis
C. ELISA
1. Aspergillus
D. EIA
1. Blastomyces
2. Candida
E. Latex agglutination
1. Cryptococcus (more sensitive than India Ink Stain in CSF)
2. Candida
F. Fluorescent antibody
1. Pneumocystis
VIII. Molecular Diagnosis of Fungal Disease
A. Probes
1. Used to identify:
a. Histoplasma capsulatum
b. Blastomyces dermatitidis
c. Coccidioides immitis
d. Cryptococcus neoformans
IX. Antifungal Susceptibility Testing
A. Appropriateness
1. CLSI has released 3 methods for fungal testing
a. Yeast testing
b. Mould testing
c. Disk diffusion testing (microtiter and Etest)
2. Concerns
a. Lack of established breakpoints for most fungal agents
b. Emergence of antifungal resistance
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 18
B. Anti-fungal classes and agents
1. Polyenes
a. Amphotericin B (primary antifungal agent used today)
2. Azoles
a. Fluconazole (primary antifungal agent in treating yeast infections)
b. Intraconazole
c. Voriconazole
3. Candins
a. Caspofungin
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology
Page 19
Basics of Mycology – Study Questions
1. True or False. Fungi resemble plants in that both contain chlorophyll.
2. True or False: Arthroconidia reproduce by fragmentation.
3. The two growth forms of fungi include:
a. Yeast and hyphae
b. Yeast and mould
c. Blastoconidia and mould
d. Conidia and hyphae
4. A mass of branching intertwined hyphae forming a matt of growth is known as:
a. Hyphae
b. Conidia
c. Mycelium
d. Favic chandeliers
5. Blastoconidia that have elongated are known as:
a. Hyphae
b. Pseudohyphae
c. Arthroconidia
d. Racquet hyphae
6. The India ink stain is used to detect what part of a yeast cell?
a. Chlamydoconidia
b. Pseudohyphae
c. Blastoconidia
d. Capsule
7. A fungus that has a yeast phase that grows at 35°C and a mold phase that grows at 30°C is
referred to as:
a. Dimorphic
b. Polymorphic
c. Bimorphic
d. Anamorphic
8. The purpose for using a KOH preparation when doing a direct examination for fungi is to:
a. Dissolve the keratin to unmask the fungus elements
b. Stain the hyphae and conidia of the fungi
c. Reveal capsules that may be found around yeast cells
d. Kill any bacteria that may be present in the specimen
9. In the Lactophenol aniline blue stain, what reagent stains the chitin in the fungal cell wall?
a. Aniline blue
b. Phenol
c. Lactic acid
d. Glycerol
10. What is the atmospheric requirement for fungi to grow?
a. Anaerobic
b. Ambient air
c. 5-10% CO2
d. >10% CO2
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology – Case Studies & Study Questions
Page 20
11. Of the following techniques, which one is the best for maintaining the juxtaposition of the fungal
elements and can be kept in the laboratory permanently?
a. Tease preparation
b. Scotch tape preparation
c. Slide culture
12. Which of the following approaches is NOT used in the identification of fungi?
a. Growth rate
b. Colony morphology
c. Microscopic appearance
d. Methylene blue stain
13. What is/are the benefits to performing a direct mount examination on every specimen submitted
for fungal culture?
a. Allows you to send out an immediate preliminary report to the primary care provider so they
can initiate treatment or look for other diagnoses
b. Enables you to know if special media needs to be inoculated
c. Allows you to observe the yeast phase of dimorphic organisms
d. May provide a clue as to the identity of the causative agent without having to wait for the
fungus to incubate
e. All of the above
14. Match the media with its general use (answers can be used more than once and items can have
more than one answer):
_____ Mycosel agar
a. Nonselective, primary isolation media
b. Demonstrate phenol-oxidase enzyme
c. Promotes blastoconidia, chlamydospores and
hyphal formation
d. Selective media
e. Stimulates spore formation and pigmentation
f. Used to demonstrate yeast phase of dimorphic
fungi
_____ Sabouraud’s Dextrose agar
_____ Brain Heart Infusion agar
_____ Cornmeal Tween 80 agar
_____ Inhibitory Mold agar
_____ Niger Seed agar
_____ Potato Dextrose agar
15. Match the image with its descriptive term.
_____ Mycelium
a.
d.
_____ Nodular organ
_____ Spiral hyphae
b.
e.
_____ Racquet hyphae
_____ Favic chandelier
CLS419 Clinical Microbiology II
Rotation II
c.
Mycology
Basics of Mycology – Case Studies & Study Questions
Page 21
16. Match the picture of the asexual structure with its descriptive term.
_____ Blastoconidia
_____ Arthroconidia
a.
d.
_____ Macroconidia
_____ Microconidia
b.
_____ Phialoconidia
e.
_____ Sporangiospores
c.
17. List three general rules for good collection of fungal specimens.
18. A nurse calls you and asks how to collect a toenail sample for a fungal culture. Give complete
instructions that you would give to the nurse. Then describe a basic outline of what process you
as a clinical laboratory scientist will follow relating to direct examination, specimen processing
and fungal culture setup when you receive the specimen in the lab.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Basics of Mycology – Case Studies & Study Questions
Page 22
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Three:
Yeast
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast
Page 23
YEAST
Objectives:
Upon completion of this CLS 419 unit, the Clinical Laboratory Science student will:
1.
Describe the following characteristics common to yeast:
a. Gram stain morphology
b. Colony morphology
c. Growth rate
2.
Describe the following structures seen in yeasts:
a. Blastoconidia
b. Arthroconidia
c. Chlamydoconidia
d. Germ tube
e. Capsule
f.
Pseudohyphae
3.
Explain the principle of the following tests, including the interpretation of results:
a. Germ tube production
b. Antigen testing
c. Urease
d. Niger seed agar (TOC)
e. Carbohydrate utilization
4.
a.
b.
c.
d.
e.
Differentiate Candida albicans from Candida glabrata, based on the following:
Cornmeal-Tween-80 agar
Germ tube
Rapid trehalose assimilation
Colony morphology on Sheep Blood Agar
Gram stain/wet prep
5.
Describe the specific growth requirements of Malassezia furfur.
6.
Correlate the clinical, epidemiological and laboratory findings associated with the following
infections:
a. Candida albicans
1) Cutaneous (diaper rash, onychomycosis)
2) Mucocutaneous (thrush, vaginitis, pulmonary and alimentary infections)
3) Systemic (UTI, diabetics, endocarditis)
b. Candida species
1) Opportunistic infections
c. Cryptococcus neoformans
1) Respiratory infections
2) Meningitis
d. Candida glabrata
1) Urinary tract infections
e. Malassezia furfur
1) Tinea versicolor
2) Systemic infections associated with nutritional lipid supplements
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast
Page 24
YEAST
I.
General Characteristics
A. Can isolate from almost any specimen
1. Generally considered normal flora
2. Opportunistic pathogen
B. Unicellular
C. Reproduction
1. Asexual
a. Blastoconidia (budding yeast)
 Pseudohyphae = elongated blastoconidia
b. Arthroconidia
2. Sexual
a. Ascospores (acid fast positive = Saccharomyces
species)
D. Direct examination
1. Observe for reproductive structures
2. Gram stain: large, gram-positive budding yeast and pseudohyphae
2. India ink prep: observe for capsules around cells (Cryptococcus)
E. Growth
1. Rate: 2-3 days on Sheep Blood Agar and most other nonselective primary isolation
media
2. Temperature: 22-37°C (best at 30°C)
F. Colony morphology
1. Appearance
a. Similar to bacterial colony
b. Moist, smooth
2. Texture
a. Can be glabrous (waxy), mucoid, butter-like, wrinkled, or velvety
b. Can see filaments around perimeter of colony
3. Color
a. White, cream, tan (rarely pink or salmon)
4. May be confused with Staphylococcus species
a. Yeast grow on sheep blood agar
b. Yeast are catalase positive and coagulase negative just like Staph epi
c. MUST confirm with Gram stain or LPAB stain
II. Yeast Identification Methods
A. Germ Tube Production
1. First test performed upon isolation of yeast
2. Most important and easiest test to perform to identify yeast
3. Procedure
a. Emulsify small amount of yeast in tube containing 0.5-1.0 ml sterile serum (rabbit,
fetal calf, or human)
b. Incubate at 35°C for 2.5-3.0 hours
c. Place one drop of suspension on a slide, coverslip, and examine microscopically on
40x for long tube-like projections (germ tubes) extending out from the yeast cells.
d. Do not confuse germ tube with pseudohyphae (constricted at point of attachment)
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast
Page 25



Germ Tube
Parallel sides
Non-septate
No constriction at point of attachment



Pseudohyphae
Not necessarily parallel
May be septate
Constricted at point of attachment
B. Microscopic morphology on Cornmeal-Tween 80 agar (CMT)
1. When germ tube is negative, subculture yeast to CMT agar
2. Refer to “Basics of Mycology” lecture (p. 15) for procedure
3. Observe microscopically for characteristic features of chlamydoconidia, blastoconidia,
pseudohyphae and arthrospores
C. Niger Seed Agar (Birdseed agar)
1. Observe for pigment production
2. Cryptococcus neoformans produces dark brown colonies on agar due to phenol oxidase
activity which decomposes melanin in yeast cell walls.
D. Biochemical Tests
a. Urease production
 Positive: Cryptococcus sp. (rapid), Trichosporon sp., Rhodotorula sp.
 Negative: Candida sp., Geotrichum sp.
b. Carbohydrate assimilation
 Ability of yeast to utilize a particular carbohydrate as a sole source of carbon in the
presence of oxygen
 Look for growth or no growth
c. Carbohydrate fermentation
 Ability of yeast to utilize a carbohydrate anaerobically
 Byproducts are alcohol and CO2, look for gas
d. Commercial identification systems
 Manual kit method examples include API 20C, API YeastIdent
 Automated method examples include MicroScan Rapid Yeast Ident, Vitek 2 YST card
III. Clinically Significant Yeast
A. Candida albicans and other Candida species
1. Normal flora of skin, mucous membranes and intestinal tract
2. Leading opportunistic fungal infection in man
a. Candidiasis (Candidosis, Moniliasis)
 Mucocutaneous (most common)
o Thrush (oral)
o Vaginitis
o Bronchial and pulmonary
o Alimentary
o Chronic mucocutaneous
 Cutaneous
o Areas of profuse sweating or immersion in water
o Skin – severe diaper rash
o Nails - oncychomycosis
 Systemic
o Most devastating
o UTI
o Endocarditis
o Septicemia
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast
Page 26
b. Factors predisposing to infection
 Immunocompromised
 Malignancy
 Prolonged antibiotic therapy
 Lupus
 Diabetes
 Pregnancy and use of birth control pills
 Age – infancy and old age
 Damaged skin barrier – catheters, burns, wounds
c.
Etiologic agents
 Candida albicans – number one fungal isolate in laboratory
 Other medically important species that can cause opportunistic infections:
o Candida (Torulopsis) glabrata
o Candida parapsilosis
o Candida tropicalis
o Candida krusei
o Candida stellatoidea
3. Identification
a. Candida albicans
 Germ tube: positive within 3 hours (95% of time)
 CMT: Clustered blastoconidia at septa of pseudohyphae,
terminal chlamydoconidia
 CHO assimilation: sucrose positive
b. Candida stellatoidea
 Germ tube: positive within 3 hours
 CMT: Blastoconidia, pseudohyphae, terminal
chlamydoconidia
 CHO assimilation: sucrose negative
c. Candida (Torulopsis) glabrata
 Second most common cause of urinary yeast infections
 Germ tube: negative at 3 hours
 CMT: Only blastoconidia, no pseudohyphae
 CHO assimilation: only glucose and trehalose positive
d. Candida tropicalis
 Germ tube: rarely positive in 3 hours
 CMT: Sparse single or short-chained blastoconidia
anywhere along pseudohyphae, rare chlamydospores
 CHO assimilation: sucrose positive
e. Candida krusei
 Germ tube: negative at 3 hours
 CMT: Tree-like branching of abundant blastoconidia from
the septa of elongated pseudohyphae (“crossmatchsticks” appearance)
f. Candida parapsilosis species complex
 Germ tube: negative at 3 hours
 CMT: Few single or small clustered blastoconidia at or
between septa of thin curved pseudohyphae. Sometimes
giant pseudohyphae may be observed
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast
Page 27
B. Cryptococcus neoformans
1. Causes Cryptococcosis
a. Inhale yeast and develop mild respiratory infection
b. Primary pulmonary disease – debilitated patients, overwhelming or continual
exposure - pneumonia
c. Meningitis – organism has predilection for brain and meninges
d. Septicemia
e. Major cause of opportunistic infection in AIDS patients
2. Isolated from soil contaminated by pigeon droppings (major source), fruits, milk, plants,
feces of normal humans
3. Identification
a. Direct examination
 Gram stain: very large and round budding yeast, may not stain well if
polysaccharide capsule present
 India ink stain
o Positive 50% of time in CSF
o Aids in viewing polysaccharide capsule
b. Colony morphology – mucoid due to capsules
c. Germ tube: negative at 3 hours
d. CMT: large, round blastoconidia, no pseudohyphae
e. Urease: rapidly positive (within 3 hours)
f. Niger seed agar, TOC agar
 Cryptococcus neoformans: brown pigment
 Other Cryptococcus species and other yeasts: no brown pigment
g. Serological testing
 Cryptococcal antigen test
o Indirect agglutination test
o Antibody to polysaccharide capsule is coated onto latex particles
o Test for antigen (capsule) in CSF and serum
o More sensitive than India Ink prep
C. Geotrichum candidum
1. Causes Geotrichosis – pulmonary disease in immunocompromised patients
2. Disease state is similar to Candida – often pulmonary or mimics thrush (mucocutaneous)
3. Not a true yeast, is really a mold
4. Identification
a. Germ tube: negative at 3 hours
b. CMT: arthroconidia and true hyphae
c. Urease: negative
D. Malassezia furfur
1. Causes Tinea versicolor (Pityriasis versicolor)
a. Superficial fungal infection
b. Infection of the horny layer of the epidermidis
c. Characterized by reddish-brown, brown or white scaly patches on skin
d. Lesions fluoresce yellow under Wood’s lamp
2. Also can cause septicemia in patients receiving lipid therapy by IV
3. Identification
a. KOH preparation of skin scrapings: clusters of thick-walled,
round budding yeast and short, straight or angular mycelial
fragments (spaghetti and meatball appearance)
b. Lipophilic: in order to culture organism, must overlay culture
media with olive oil (growth occurs in 24 hours)
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast
Page 28
Yeast Case Study #1
A 45-year old pigeon breeder, who recently received a renal transplant (and thus was on
immunosuppressive steroids), complained to his physician of headache, dizziness, blurred vision, and
a stiff neck. Specimens of purulent cerebrospinal fluid were sent to the microbiology laboratory. A
carefully examined Gram stain of the CSF sediment was negative; however, on brain heart infusion
agar with blood at 30ºC, a white, mucoid yeast rapidly grew. The organism was inhibited on brain
heart infusion agar with blood, gentamicin, cyclohexamide and chloramphenicol.
1. Which disease do you suspect? Circle the letter of the correct answer.
a. Candidiasis
b. No disease – organism was a laboratory contaminant
c. Cryptococcosis
d. Histoplasmosis
2. Give three reasons for choosing your answer to question #1.
3. If the yeast was significant, why was it not observed on the CSF Gram stain?
4. If your answer to question #1 was not “b”, what three tests would you perform to speciate the
yeast?
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast – Case Studies & Study Questions
Page 29
Yeast Case Study #2
Directions - Within CLS419 Clinical Microbiology II, go to Assignments, Clinical Rotation – Mycology,
and then Yeast Unit. Go to the corresponding case study to view the microscopic examples for each
case. Click on an image to see an enlarged view. Answer the following short answer or single answer
multiple choice questions for each case.
This organism was isolated from the sputum of a HIV+, 37-year-old male with a CD4 cell count of
<200/mm3. The organism grew on SDA (25ºC), and blood & chocolate agar (35ºC), at 24 hours of
incubation.
1. Presumptively identify the fungus.
2. Which of the following conventional tests should be performed to confirm the identification?
A. Germ tube production, slide culture
B. Germ tube production, urease production
C. Phenol oxidase activity (Birdseed or Niger seed agar), urease production
D. Slide culture, urease production
Due to the identification of the fungus in the patient’s sputum, a CSF was submitted for direct antigen
testing. Look at the test results on Blackboard.
The test utilizes two latex suspensions:
Antigen latex – detects polysaccharide capsule
Normal globulin latex – detects interfering substances in specimen which cause a false +
reaction
Each specimen and control is tested with both latex suspensions, except for the ‘normal globulin
positive control which is tested only with the normal globulin latex.
3. The direct antigen test results indicate the patient’s CSF is:
A. Negative for antibodies to the polysaccharide capsule
B. Negative for the polysaccharide capsule
C. Positive for antibodies to the polysaccharide capsule
D. Positive for the polysaccharide capsule
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast – Case Studies & Study Questions
Page 30
Yeast Study Questions
1. Match the yeast with its key identifying characteristic:
____
Candida parapsilosis
a. Short-chained blastoconidia anywhere
along pseudohyphae
b. Thick capsule
c. Cross-matchsticks blastoconidia
d. Terminal chlamydospores
e. Giant pseudohyphae
____
Candida krusei
____
Candida tropicalis
____
Candida albicans
____
Cryptococcus neoformans
2. Identify the structures shown in these two drawings.
a. ____________________
b. ____________________
c.
____________________
d. ____________________
D
3. What key test is used to definitively identify Candida
albicans?
a. Gram stain
b. Germ tube
c. Colony morphology
d. KOH preparation
4. The number 1 fungal agent isolated in the clinical laboratory is:
a. Malassezia furfur
b. Cryptococcus neoformans
c. Candida albicans
d. Candida glabrata
5. What disease(s) does Candida albicans cause?
a. Mucocutaneous infections
b. Cutaneous infections
c. Systemic infections
d. All of the above
6. Which yeast, in order to grow on culture media, requires olive oil to be overlaid the media?
a. Malassezia furfur
b. Cryptococcus neoformans
c. Candida albicans
d. Candida glabrata
7. True or False: All Candida albicans produce germ tubes and chlamydospores.
8. True or False: A germ tube is constricted at its point of attachment to the mother cell.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast – Case Studies & Study Questions
Page 31
9. Which yeast can be identified when the germ tube is negative and carbohydrate assimilation of
glucose and trehalose?
a. Cryptococcus neoformans
b. Candida albicans
c. Candida stellatoidea
d. Candida glabrata
10. What are pseudohyphae?
11. What steps are taken to identify yeast in the clinical laboratory?
CLS419 Clinical Microbiology II
Rotation II
Mycology
Yeast – Case Studies & Study Questions
Page 32
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Four:
Opportunistic Molds
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds
Page 33
OPPORTUNISTIC MOULDS
Objectives:
Upon completion of this CLS 419 unit, the Clinical Laboratory Science student will:
1.
Discuss the following characteristics common to opportunistic molds:
a. Rapid growth
b. Airborne
c. Opportunistic pathogens
d. Frequent laboratory contaminants
2.
Discuss the following characteristics common to the Zygomycetes:
a. Asexual reproductive structures
b. Aseptate hyphae (sparsely septate)
c. Mycelial growth
3.
Identify the following fungi using macroscopic and microscopic morphology:
a. Absidia species
b. Mucor species
c. Rhizopus species
d. Aspergillus fumigatus
e. Aspergillus flavus
f.
Aspergillus niger
g. Penicillium species
h. Fusarium species
i.
Alternaria species
4.
Correlate the clinical, epidemiological and laboratory findings associated with the following
infections:
a. Zygomycetes
1) Zygomycosis
2) Predisposing conditions (diabetes)
b. Aspergillus fumigatus/Aspergillus flavus
1) Aspergillosis
a) Allergic bronchopulmonary (farmer’s lung)
b) Colonizing (fungus ball)
c) Invasive forms
c. Aspergillus niger
1) Swimmer’s ear
d. Fusarium species
1) Mycotic keratitis
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds
Page 34
OPPORTUNISTIC MOLDS
I.
Basic Characteristics
A. Rapid growers – initial growth at 2-3 days with mature colonies in 4-5 days
B. Found in soil (saprobic) and conidia/spores are airborne
C. Opportunistic pathogens
1. Normally inhaled – dependent upon body’s response after inhalation
2. Immunocompromised patients are susceptible (AIDS, cancer, transplant)
D. Treatment
1. Can be very toxic
2. Important to determine if isolate is a contaminant or causing disease
3. Examine tissue or isolate from more than one body site to determine pathogenesis
II. Aseptate (sparsely septate) opportunistic molds
A. Zygomycetes
1. Identification
a. Colony morphology
 Lid lifters
o Mycelium very cottony (wooly)
b. Microscopic examination
 Asexual reproductive structures
o Sporangiospores inside sporangium
o Broad, irregular width, ribbon-like aseptate
hyphae
o Rhizoids
2. Clinical Significance
a. Causes Zygomycosis
b. Predisposition
 Diabetics in ketoacidosis
 Malnutrition
 Immunosuppressed
 Prolonged antibiotic therapy (decrease normal flora)
c. Pathogenesis
 Inhale spores
 Infects nasal sinuses (nasal discharge is black)
 Spreads to adjacent blood vessels
 Causes necrosis and vascular thrombosis (blood clots)
 Can migrate to brain and meninges which can lead to rapidly fatal
meningoencephalitis (2-10 days after infection)
3. Examples
a. Absidia species
 Colony morphology – cottony texture, initially white
becoming gray to gray-brown
 Microscopic – Internodal rhizoids (root-like hyphae)
b. Mucor species
 Colony morphology – cottony texture, initially dirty-white
becoming mousy-brown to gray
 Microscopic – No rhizoids
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds
Page 35
c.
Rhizopus species
 Most common zygomycete causing human disease
 Colony morphology – cottony texture, initially white
becoming grey to brown
 Microscopic – Nodal rhizoids
II. Septate opportunistic molds
A. Dematiaceous hyphae
1. Hyphae and/or conidia are dark colored due to melanin-like pigment with septations
2. Colonies are dark gray, brown, or black, wooly, hairy, or velvety surface with smoky gray
to jet black on reverse
3. Identification of genus/species is accomplished by microscopic examination
a. Dark yellow-brown mycelium composed of uniform hyphae with parallel walls and
distinct sepations
4. Example
a. Alternaria species
 Colony morphology: shades of gray to brown to black,
texture is downy to cottony
 Microscopic exam: short chains of large, smoothwalled, multicelled, macroconidia separated by both
cross and longitudinal septa. Macroconidia are drumstick-shaped with elongated
beak of one conidium butting against the rounded blunt end of the next.
 Pathogenesis: Chronic fungal sinusitis, can become systemic in
immunosuppressed patients
B. Hyaline hyphae
1. Hyphae and/or conidia are light colored with septations
2. Identification of genus/species is accomplished by microscopic examination
3. Examples
a. Aspergillus species
 Second most isolated fungus in the clinical laboratory
 Causes Aspergillosis
o Predisposed: debilitated and immunocompromised patients
o Pathogenesis: inhale conidia  develop sinusitis or bronchopulmonary
disease  can invade surrounding blood vessels  becomes systemic
(acute and fatal). It is very invasive and has a rapid process for spreading.
o Can also be non-invasive in form of fungal ball and allergic fungal infections
o Most susceptible include bone marrow and transplant recipients, hematologic
malignancies, and immunocompromised (90% mortality rate)
 Identify by evaluating colony morphology (distinct outer margin with a white
apron at the advancing area of growth) and microscopic arrangement of conidia
on the phialides and vesicle to speciate

Aspergillus fumigatus
o Most common pathogenic Aspergillus species
o Colony morphology: Initially white, eventually
turning some shade of blue-green, green-gray, or
green-brown; texture: granular to cottony
o Microscopic exam: Single row of phialides on the
top half of a club-shaped vesicle producing long
chains of spherical conidia that tend to bend
inward towards a center axis. Conidiophore is long,
smooth-walled, and derives from a foot cell.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds
Page 36

Aspergillus niger
o Can cause swimmer’s ear
o Colony morphology: Initially white to yellow, then
turning black (salt and pepper appearance) due to jetblack conidia, reverse is buff or yellow-gray (rules out
dematiaceous), texture is cottony
o Microscopic exam: Double row of phialides covering
entire vesicle (radiate head). Single and short chains
of conidia extend from phialides and when mature are
roughened. Conidiophore is smooth-walled and
derives from a foot cell.

Aspergillus flavus
o Colony morphology: some shade of yellow, yellowgreen, or yellow-brown; texture is granular to cottony
o Microscopic exam: Single or double row of phialides
cover entire vesicle radiating in all directions.
Conidiophore is long with distinct roughening of wall
proximal to junction of vesicle (when mature) and
derives from a foot cell.
b. Penicillium species
 Colony morphology: initially velvety and white, as matures
becomes granular with various shades of green or bluegreen (yellow and yellow-brown can also be seen)
 Microscopic exam: conidiophores branch into metulae and
then phialides (tips are blunt), from the tips of the phialides
emerge chains of spherical conidia (“brush-like” or “skeleton
fingers”)
c.
Fusarium species
 Most common cause of mycotic keratitis
 Colony morphology: Woolly or cottony, initially white and
later becomes lavender, rose-red, or magenta on surface
and reverse
 Microscopic exam: Macroconidia are 2-5 celled (separated
by traverse septa), sickle-shaped (“canoes”) with a
distinctive foot cell at the point of attachment
d. Sepedonium species
 Must distinguish from Histoplasma capsulatum, a dimorphic fungus
 Colony morphology: Waxy and white colony rapidly
becomes velvety with lemon color having a peripheral
fringe, and white reverse.
 Microscopic exam: Single or clustered, thick-walled,
smooth to rough macroconidia at the ends of simple or
branched conidiophores
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds
Page 37
Opportunistic Mold Case Studies
Directions - Within CLS419 Clinical Microbiology II, go to Assignments, Clinical Rotation – Mycology,
and then Opportunistic Mold Unit. Go to the corresponding case study to view the microscopic
examples for each case. Click on an image to see an enlarged view. Answer the following short
answer or single answer multiple choice questions for each case.
Case 1
This organism was isolated from blood-tinged, nasal discharge of a diabetic, 65-year-old male.
Growth was detected after 2 days of incubation at 25ºC on Sabouraud dextrose agar (SDA), with no
growth on Mycosel agar.
1. Identify this fungus.
2. This class of fungus has __________ hyphae and an asexual reproductive structure called a
___________________.
A. Aseptate/sparsely septate, sporangium
B. Dematiaceous, septate
C. Hyaline, rhizoid
D. Septate, phialide
3. Macroscopically, this fungus’ colony is described as:
A. Rapidly growing, flat, velvety
B. Rapidly growing, wooly to cottony
C. Slow growing, yeast-like
D. Slow growing, granular to powdery
4. This class of fungi is the causative agent of ________________which is associated with infection
of the __________________________.
A. Aspergillosis, lower-respiratory tract presenting as allergy, colonization or systemic infections
B. Chromoblastomycosis, upper-respiratory tract which may become systemic
C. Dermatophytosis; hair, skin and nails
D. Zygomycosis, nasal sinuses spreading to the eyes and brain via blood vessels
Case 2
This fungus was isolated from the lung of a 35-year-old female lymphoma patient. Growth was
detected after 3 days of incubation at 25ºC on SDA, with no growth on Inhibitory mold agar.
1. Identify this fungus.
2. Microscopic characteristics of this fungus include:
A. Dematiaceous septate hyphae, phialides radiate from vesicle
B. Hyaline aseptate/sparsely septate hyphae, sporangiums
C. Hyaline septate hyphae, macroconidia produce phialoconidia described as a “paint brush”
D. Hyaline septate hyphae, phialides cover upper 2/3 of vesicle
3. Some species of this genus are the etiologic agents of:
A. Aspergillosis, presenting as allergy, colonization or systemic infections
B. Chromoblastomycosis, presenting as an upper-respiratory tract infection which may become
systemic
C. Dermatophytosis; an infection of hair, skin and nails
D. Opportunistic, subcutaneous fungemia
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds – Case Studies & Study Questions
Page 38
Case 3
This fungus was isolated from the sputum of a 35-year-old male. A brown colony was detected at 72
hours of incubation at 25ºC on SDA, with no growth on Mycosel agar.
1. Identify this fungus.
2. Microscopic characteristics of this fungus include:
A. Dematiaceous aseptate/sparsely septate hyphae, chaining microconidia
B. Dematiaceous septate hyphae, chaining macroconidia w/cross and longitudinal septa
C. Hyaline septate hyphae, microconidia radiate from vesicle
D. Hyaline septate hyphae; multicelled conidia
3. Clinically, this fungus is considered to be a/an:
A. Contaminant, possible association with allergies and superficial mycosis
B. Etiologic agent of subcutaneous mycosis
C. Primary cause of upper-respiratory tract infections & mycotic keratitis
D. Primary cause of upper-respiratory tract infections & swimmer’s ear
Case 4
This organism was isolated from an inflamed cornea of a 14-year-old male contact lens wearer.
Growth was detected on SDA on day 3 of incubation at 25ºC with no growth on Mycosel agar.
1. Identify this fungus.
2. Clinically, this fungus is considered to be a/an:
A. Contaminant, non-pathogenic
B. Contaminant, possible association with allergies and superficial mycosis
C. Etiologic agent of subcutaneous mycosis
D. Primary cause of mycotic keratitis & also fungemia w/disseminated infection in
immunocompromised
3. A medical student submitted to the laboratory a Mycosel slant inoculated at bedside with corneal
scrapings he had collected from a 20-year-old female inpatient. He indicated that the physician
was trying to rule out mycotic keratitis. What is the next best step?
A. Incubate the slant at 35 C for two weeks.
B. Reject the culture as corneal scrapings are inappropriate specimens to culture
C. Request another scraping be performed in order to inoculate a primary isolation media
without cycloheximide
D. Request another scraping be performed in order to inoculate Lowenstein-Jensen and ReganLowe media
Case 5
This organism was isolated from the sputum of a 50-year-old female who has smoked an average of
1/2 to 1 pack of cigarettes per day for 25 years. Growth was detected on SDA on day 3 of incubation
at 25ºC with no growth on SDA w/cycloheximide & antibiotics.
1. Identify the fungus.
2. Clinically, this fungus is considered to be a/an:
A. Contaminant, rarely an opportunistic pathogen
B. Etiologic agent of subcutaneous mycosis
C. Infectious agent often presenting as allergy, colonization or systemic infections
D. Primary cause of upper and lower-respiratory tract infections
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds – Case Studies & Study Questions
Page 39
Opportunistic Mold Study Questions
1. Match the drawing of a zygomycete with the name of the structure.
_____ Hyphae or Stolon
_____ Rhizoid
_____ Sporangium
2. Match the drawings of the zygomycete with their genus name.
_____ Absidia
_____ Mucor
_____ Rhizopus
A.
B.
C.
3. Which of the following possess conidia with vertical and horizontal cross-walls?
a. Alternaria species
b. Cladosporium species
c. Fusarium species
d. Sepedonium species
4. Key characteristic(s) of the opportunistic molds include:
a. Frequent lab contaminants
b. Rapid growers
c. Grow at 25-30°C
d. All of the above
5. A key characteristic about the colony morphology of the Zygomycetes is:
a. Black pigmentation
b. Granular texture
c. Peppered appearance
d. Lid lifters
6. A key characteristic about the colony morphology of Aspergillus niger is:
a. White pigmentation
b. Granular texture
c. Peppered appearance
d. Lid lifters
7. Which opportunistic mold can cause farmer’s lung (fungal ball), allergic reactions, sinusitis, and
become invasive to affect multiple organ systems?
a. Aspergillus fumigatus
b. Aspergillus niger
c. Fusarium species
d. Rhizopus species
8. True or False: Opportunistic molds will grow on Mycosel agar.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds – Case Studies & Study Questions
Page 40
9. Complete the table:
Mold
Hyphae appearance
(Septate / Sparsely septate)
Hyphae color
(Dematiaceous / Hyaline)
Mucor species
Rhizopus species
Aspergillus species
Alternaria species
Penicillium species
CLS419 Clinical Microbiology II
Rotation II
Mycology
Opportunistic Molds – Case Studies & Study Questions
Page 41
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Five:
Dermatophytes
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes
Page 42
DERMATOPHYTES
Objectives:
Upon completion of this CLS 419 unit, the Clinical Laboratory Science student will:
1.
Define the clinical term “tinea”.
2.
Differentiate Trichophyton mentagrophytes and Trichophyton rubrum based on the urease and in
vitro hair penetration reactions.
3.
Correlate the clinical, epidemiological and laboratory findings, including macroscopic and
microscopic morphology and growth rate associated with the following organisms:
a. Microsporum canis
b. Microsporum gypseum
c. Microsporum audouinii
d. Epidermophyton floccosum
e. Trichophyton mentagrophytes
f.
Trichophyton rubrum
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes
Page 43
DERMATOPHYTES
I.
Characteristics
A. Epidemiology – worldwide distribution
1. Anthropophilic – found primarily in humans
2. Zoophilic – found primarily in animals such as cats and dogs (man easily infected)
3. Geophilic – found primarily in soil
B. Clinical Significance
1. Causes Tinea (Latin for “worm” or “ringworm”), also referred to as dermatophytoses –
infections that involve the superficial areas of the body, including the hair, skin and nails
2. Dermatophytes break down and utilize keratin as a source of nitrogen (but unable to
penetrate the subcutaneous tissue)
3. Body areas affected
a. Skin
i. Tinea corporis (ringworm of the body) – any and all common dermatophytes may
be involved in this type of infection
ii. Tinea pedis (ringworm of the feet) – athlete’s foot
iii. Tinea cruris (ringworm of the groin area) – jock itch
b. Hair
i. Tinea capitis (ringworm of hair and/or scalp)
o Ectothrix hair invasion – chains of arthroconidia outside the hair shaft
ii. Tinea barbae (ringworm of beard hair)
o Ectothrix hair invasion – chains of arthroconidia outside the hair shaft
iii. “Black dot” ringworm
o Endothrix hair invasion – chains of arthroconidia inside the hair shaft
iv. “Favic” ringworm
o Endothrix hair invasion – hyphae rather than conidia invade hair shaft
c. Nails
i. Tinea unguium (ringworm of the nails)
Dermatophyte
Microsporum
Trichophyton
Epidermophyton
Skin
X
X
X
Hair
X
X
Nails
X
X
C. Relatively rapid growing molds that can be distinguished by certain differences in colonial
morphology and in the relative production of macroconidia and microconidia. In common
practice, six species of dermatophytes cause more than 98% of human dermatophytoses.
II. Laboratory Diagnosis
A. Specimen collection
1. Hair
 Hair should be plucked, not cut, from the edge of the lesion.
 Choose hairs that fluoresce under a Wood's lamp or, if none fluoresce, choose
broken or scaly ones.
 Place infected hair into Petri dish
2. Skin
 Skin should be washed well and then scraped from the margin of the lesion onto
folded black paper
 Place skin scrapings into Petri dish
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes
Page 44
3. Nails
 Nail scrapings are obtained from the nail bed or from infected areas after the outer
layers are discarded
 Place scrapings of infected nails into Petri dish
B. Direct examination of clinical material
1. 10% KOH preparation
a. For hair, skin and nails
b. Detect presence of fungal elements
2. Calcofluor white stain
a. For hair, skin and nails
b. Detect presence of fungal elements
3. Wood’s Lamp
a. For hair only
b. Positive – bright yellow-green fluorescence
 Microsporum canis
 Microsporum audouinii
c. Negative – no fluorescence
 Microsporum gypseum
 Trichophyton species
C. Culture of clinical material
1. Specimen processing
a. Nails are scraped or minced into small pieces
b. Hair is cut into short segments
2. Media
a. Each specimen is divided between at least two types of culture media
b. The use of antibiotics will inhibit the overgrowth of bacteria and incorporation of
cycloheximide will prevent the overgrowth of the rapidly growing saprophytic fungi
c. Brain heart infusion (BHI) or Sabouraud’s brain heart infusion agar
 General purpose fungal media
d. Mycosel
 Good growth – clue that may have a dermatophyte
3. Growth requirements
a. Generally 25-30°C
 Exception: Trichophyton verrucosum requires 35°C
b. Ambient air
c. Visible growth can occur as soon as 3-4 days, with mature growth between 1-3
weeks
d. Cultures must be examined frequently for 4 weeks
D. Identification
1. Growth rate – 1-3 weeks (medium rate)
2. Colony morphology
3. Microscopic morphology: Hyaline, septate hyphae, presence or absence of macroconidia
and microconidia
a. Scotch tape preparation and tease prep - quick way to identify at least to genus level
b. Slide culture
 Use Potato dextrose and Cornmeal dextrose
o Encourages sporulation
o Enhances pigment production
 Incubate at least 7 days
 Able to get to the Genus level of ID; possibly species level
 Many labs will just report to the Genus level
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes
Page 45
4. Physiologic tests
a. Able to speciate the fungi
b. Urea hydrolysis
 Incubate 2-3 days on Christensen’s Urea Agar
 Used to differentiate between Trichophyton rubrum (negative) and Trichophyton
mentagrophytes (positive)
c. Hair perforation test
 Sterile hair is incubated at 25ºC with mold for 10-14 days in moist chamber
 Observe hair microscopically for presence or absence of conical perforations of
the hair shaft
 Used to differentiate between Trichophyton rubrum (negative) and Trichophyton
mentagrophytes (positive)
d. Growth on rice grain media
 Microsporum audouinii will not grow
 All other Microsporum species will grow
e. Vitamin requirements
 Some Trichophyton species require vitamins (thiamine and inositol) to grow
 Trichophyton verrucosum and T. violaceum
III. Etiologic agents
A. Microsporum species
1. Clinical Significance
a. Invades hair and skin only
2. Microsporum canis
a. Colony morphology
i. Growth at 3-5 days (rapid)
ii. Flat with feathery edges; initially white and silky, later develops lemon-yellow
pigment at periphery
iii. Reverse: lemon yellow or yellow-brown
b. Microscopic examination
i. Macroconidia: echinulate (spiny surface), thick-walled, spindle-shaped, multicelled (3-15), tapering distal ends often pointed and slightly turned to one side at
tip
ii. Microconidia: rarely seen, small, hyaline, tear-drop or elliptical shape, borne
laterally directly from hyphae
c. Causes tinea capitis, tinea corporis, often from contact with dogs and cats
i. Hair will fluoresce under Wood’s Lamp
3. Microsporum gypseum
a. Colony morphology
i. Growth at 3-5 days (rapid)
ii. Flat, initially white but turning fawn-brown (cinnamon) to reddish-brown, granular
as conidia are produced
iii. Reverse: light tan
b. Microscopic examination
i. Macroconidia: moderately thick-walled, rough surface, elliptical, rounded tip, and
multi-celled (up to 6)
ii. Microconidia: few or absent
c. Causes ectothrix tinea capitis
i. Hair will NOT fluoresce under Wood’s Lamp
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes
Page 46
4. Microsporum audouinii
a. Colony morphology
i. Growth at 2 weeks (slow)
ii. Downy white to salmon-pink
iii. Reverse: tan to salmon-pink
b. Microscopic examination
i. Sterile hyphae: terminal chlamydospores, Favic chandeliers, and pectinate
bodies
ii. Macroconidia: rarely seen – bizarre-shaped
iii. Microconidia: rare or absent
c. Causes gray-patch tinea capitis in children
i. Hair will fluoresce under Wood’s Lamp
B. Epidermophyton species
1. Clinical Significance
a. Invades skin and nails only
b. Causes tinea cruris, tinea unguium
2. Epidermophyton floccosum
a. Colony morphology
i. Growth at 3-5 days (rapid)
ii. Flat, initially gray-white then develops khaki-green center that tends to be folded;
periphery is yellow to yellow-tan with feathered edges; granular as conidia
develop
iii. Reverse: yellowish brown with observable folds
b. Microscopic examination
i. Macroconidia: abundant, large, smooth thin-walled, multi-celled (2-5), clavate
(club-shaped), and borne singly or in clusters of 2-4
ii. Microconidia: absent
C. Trichophyton species
1. Clinical Significance
a. Invades hair, skin, and nails
2. Trichophyton mentagrophytes
a. Colony morphology
i. Growth at 3-10 days
ii. Varies: granular and cottony varieties; cottony type initially white, then tan,
granular type initially white or off-yellow, then tan/brown
iii. Reverse: buff to reddish brown
b. Microscopic examination
i. Macroconidia: smooth, thin walls, cigar or pencil-shaped, and multi-celled (4-5);
numerous or rare depending upon strain
ii. Microconidia: many round to globe-like (may be tear-shaped) borne in loose
grapelike clusters or laterally along hyphae, spiral hyphae in 30% of isolates
c. Causes tinea corporis, tinea capitis, tinea barbae
d. Urea hydrolysis: positive
e. Hair perforation test: positive
3. Trichophyton rubrum
a. Colony morphology
i. Growth at 2 weeks
ii. Varies from white downy to pink granular; rugal folds common
iii. Reverse: yellow when young; however, wine red color commonly develops with
age (thus named rubrum)
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes
Page 47
b. Microscopic examination
i. Macroconidia: usually absent, smooth, thin-walled, multi-celled, & pencil-shaped
ii. Microconidia: usually teardrop, most commonly borne along sides of hyphae
giving “birds on a fence” appearance
c. Causes tinea corporis, tinea pedis, tinea cruris, tinea capitis
d. Urea hydrolysis: negative
e. Hair perforation test: negative
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes
Page 48
Dermatophyte Case Studies
Directions - Within CLS419 Clinical Microbiology II, go to Assignments, Clinical Rotation – Mycology,
and then Dermatophyte Unit. Go to the corresponding case study to view the microscopic examples
for each case. Click on an image to see an enlarged view. Answer the following short answer or
single answer multiple choice questions for each case.
Case 1
This fungus was isolated from scrapings of a thickened, discolored fingernail. Growth was detected at
two weeks of incubation at 25ºC on both SDA & Mycosel agar.
1. Identify the fungus.
2. What direct exam procedure can be performed on a portion of the nail scrapings to preliminarily
determine if fungal elements are present?
A. Gram stain
B. Iodine wet preparation
C. KOH preparation
D. Modified acid-fast stain
3. Clinically, this fungus is considered to be part of the _____________ group, which is able to
infect:
A. Dermatophyte; hair, skin and nails
B. Microsporum, all tissue containing keratin
C. Opportunistic, any type of tissue
D. Subcutaneous, superficial and subcutaneous tissue
Case 2
This fungus was isolated from a seven-year-old female's "ringworm-type" lesion. After a week of
incubation at 25ºC, the organism grew on SDA & Mycosel agar exhibiting a reddish pigment on the
reverse side of the agar.
1. Identify the fungus.
A. Acremonium sp.
B. Epidermophyton sp.
C. Microsporum sp.
D. Trichophyton sp.
2. What test(s) can be utilized to speciate this organism?
A. Carbohydrate assimilation panel, phenol oxidase activity
B. Cornmeal Tween 80 agar morphology, modified acid fast staining reaction
C. Gomori-methenamine-silver nitrate and Periodic Acid-Schiff staining reactions
D. In vitro hair penetration, urea hydrolysis &/or Trichophyton Agars
Case 3
This fungus was isolated from the hair of a nine-year-old boy. The mold grew on both SAB & Mycosel
agar exhibiting a flat, granular, light tan to cinnamon colony at 5 days of 25ºC incubation.
1. Identify this fungus.
2. This genus will infect:
A. All tissue types
B. Hair, skin and nails
C. Hair and skin
D. Skin and nails
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes – Case Studies & Study Questions
Page 49
Case 4
This fungus was isolated from a small, circular skin lesion on the back of a 14-year-old boy. The mold
grew on both primary fungal isolation media, as well as media containing cycloheximide at 5 days of
25ºC incubation. The colony was irregularly shaped with a bright yellow reverse.
1. Identify this fungus.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes – Case Studies & Study Questions
Page 50
Dermatophyte Study Questions
1. Match the dermatophytes with their microscopic identifying characteristics.
Dermatophyte
____
Microsporum canis
____
Microsporum gypseum
____
Microsporum audouinii
____
Epidermophyton floccosum
____
Trichophyton mentagrophytes
____
Trichophyton rubrum
Microscopic Characteristics
a. Rare bizarre-shaped macroconidia and rare or
absent microconidia
b. Rare smooth, thin-walled, multi-celled, & pencilshaped macroconidia and many teardrop
microconidia that line up singly along hyphae
c. Many rough, thick-wall, multicelled macroconidia
that are spindle-shaped with a curved pointed
tip, and few or absent microconidia
d. Rare thin smooth-walled, multicelled
macroconidia that are pencil-shaped, and many
hyaline, spherical or teardrop microconidia in
grape-like clusters
e. Large thin, smooth walls, multi-celled
macroconidia that are club-shaped and cluster
together from hyphae, and absent microconidia
f. Many rough, moderately thick-wall, multicelled
macroconidia that are elliptical with a rounded
tip, and few or absent microconidia
2. In what ways can Trichophyton mentagrophytes and Trichophyton rubrum be differentiated from
each other?
a. Colony morphology
b. Urease production
c. Hair penetration test
d. All of the above
3. What is the purpose of the KOH preparation when examining skin scrapings?
4. Check what types of infections each dermatophyte genus can cause:
Dermatophyte
Skin
Hair
Nails
Microsporum
Trichophyton
Epidermophyton
CLS419 Clinical Microbiology II
Rotation II
Mycology
Dermatophytes – Case Studies & Study Questions
Page 51
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Six:
Subcutaneous Fungi
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 52
SUBCUTANEOUS FUNGI
Objectives:
Upon completion of this CLS 419 unit, the Clinical Laboratory Science student will:
1.
Define the following terms:
a. Chromoblastomycosis
b. Mycetoma
1)
Actinomycotic
2)
Eumycotic
c. Sulfur granules
2.
Differentiate the three types of conidia formation seen with the etiologic agents of
chromoblastomycosis:
a. Phialophora
b. Cladosporium
c. Acrotheca
3.
Differentiate between Nocardia, Streptomyces, Actinomyces and Mycobacterium species based
on:
a. Gram stain morphology
b. Acid fast stain (modified)
c. Oxygen requirements
d. Growth in/on:
1)
Non-inhibitory fungus media
2)
Blood agar
3)
Lowenstein-Jensen
4)
Thioglycollate broth
4.
Correlate the clinical, epidemiological and laboratory findings, including macroscopic and
microscopic morphology, and growth associated with the following infections:
a. Phialophora verrucosum
1)
Chromoblastomycosis
b. Cladophiliaophora carrionii
1)
Chromoblastomycosis
c. Fonsecaea species
1)
Chromoblastomycosis
d. Sporothrix schenckii
1)
Sporotrichosis
e. Acremonium species
1)
Eumycotic mycetoma
f.
Actinomyces species
1)
Actinomycotic mycetoma
g. Nocardia species
1)
Nocardiosis
5.
Differentiate Sporothrix schenckii from the opportunist Acremonium species.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 53
SUBCUTANEOUS FUNGI
I.
Basic Characteristics
A. Growth rate
1. Varies: 1-4 weeks (medium to slow rate)
B. Identification
1. Colony morphology
2. Microscopic morphology – 3 groups
a. Dematiaceous septate hyphae (majority, black molds)
b. Hyaline septate hyphae
c. Branching nocardioform bacteria
C. Clinical Significance
1. Epidemiology
a. Found in soil and on plants
b. Most are saprophytic (nonpathogenic)
2. Pathogenesis
a. Acquire by mechanical means – traumatic penetration of foreign objects such as
splinters, thorns, and sticks into the deep layers of the skin
b. Ability to elicit disease is extremely variable – depends upon:
i. Type of tissue involved
ii. Immune system of host
iii. Amount of exposure
c. Formation of a lesion at site of penetration
i. Involvement of skin, subcutaneous tissue, fascia, tendons, muscles, and bone
ii. RARELY: dissemination to visceral organs via lymphatic system
II. Laboratory Diagnosis
A. Specimen collection
1. Skin scrapings
2. Aspirated debris
3. Tissue biopsy
4. Pus/drainage from lesion
5. Sputum, BW
B. Direct examination of clinical material
1. 10% KOH preparation
a. Detect presence of fungal elements, budding yeast, sclerotic bodies, ascospores
2. Calcofluor white stain
a. Detect presence of fungal elements
3. Histological stains
a. Methenamine silver stain
b. Fontana-Masson stain
c. Hematoxylin-eosin stain
4. Gram stain
a. Detect presence of fungal elements, budding yeast, branching GPR
5. Modified acid fast stain
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 54
C. Culture of clinical material
1. Specimen processing
a. Sulfur granules must be crushed before culturing
b. Tissue grinding (when necessary)
2. Media
a. Each specimen is divided between at least two types of culture media
b. The use of antibiotics will inhibit the overgrowth of bacteria and incorporation of
cycloheximide will prevent the overgrowth of the rapidly growing saprophytic fungi
c. Sabouraud’s Dextrose Agar
 General purpose fungal media
d. Inhibitory Mold Agar
 General purpose fungal media selective for fungi
e. Mycosel Agar
 Subcutaneous molds may be inhibited by antibiotics in media
3. Growth requirements
a. Generally 25-30ºC
b. Ambient air
c. Visible growth can occur as soon as 5-10 days, with mature growth between 2-4
weeks
d. Cultures must be examined frequently for 6 weeks
D. Identification
1. Colony morphology
2. Microscopic examination
a. Scotch tape preparation and tease prep
b. Slide culture
III. Diseases and their etiologic agents
A. Chromoblastomycosis
1. Etiologic agents (black molds)
o Fonsecaea species
o Phialophora verrucosum
o Cladophialophora carrionii
2. Distribution
o Most occur in tropical and subtropical regions of world
o Occasional case in temperate regions including U.S.
o Agricultural workers most prone to infection
3. Clinical disease
o Chronic fungal infection acquired via traumatic inoculation
o Primarily involves skin and subcutaneous tissue (of mainly feet and legs)
o Develop papule at site of infection that slowly spreads to form warty or tumor-like
lesions (cauliflower-like)
o Brown sclerotic bodies, non-budding structures occurring singly or in clusters, seen in
tissues is diagnostic for chromoblastomycosis
o Secondary infection and ulceration may occur
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 55
4. Conidial structures (types of sporulation)
a. Acrotheca (rhinocladiella) type
i. Conidial heads with sympodial arrangement of
conidia, with primary conidia giving rise to
secondary conidia (looks like test tube brush)
b. Cladosporium type
i. chains of dark-staining elliptical conidia that
are branching. The conidia often show scars
(dysjunctors) at points of attachment.
Branched conidium-bearing cells appear like a
shield due to having 3 scars (note arrow in
diagram on next page)
c. Phialophora type
i. Flask-shaped phialides with collaret at tip (vase-like)
ii. Conidia – oval, one-celled, occurring at the tips of
phialides in balls (flowers in a vase)
5. Fonsecaea pedrosoi and Fonsecaea compacta
a. Colony morphology
i. Growth at 2-4 weeks
ii. Colony is brownish black, gray black or jet black in color,
texture downy
iii. Reverse: similar to surface (often jet black)
b. Microscopic examination
i. Dematiaceous (brown) septate hyphae
ii. Conidiophores cylindrical, slightly inflated at the tip
iii. Polymorphous mold – produces several types of sporulation
 Acrotheca (rhinocladiella) type sporulation
 Cladosporium type sporulation
 Phialophora type sporulation
6. Phialophora verrucosum
a. Colony morphology
i. Growth at 2-4 weeks
ii. Colony is dark gray, brown or black, velvety to wooly texture
iii. Reverse: similar to surface (often jet black)
b. Microscopic examination
i. Hyaline to dematiaceous septate hyphae
ii. Phialophora type sporulation only
7. Cladophialophora carrionii
a. Colony morphology
i. Growth at 1-4 weeks
ii. Colony is dark olive brown to brownish-black, texture is velvety
iii. Reverse: similar to surface (often jet black)
b. Microscopic examination
i. Dematiaceous septate hyphae
ii. Cladosporium type sporulation only
 Microscopic exam:
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 56
B. Mycetoma
1. Types and their etiologic agents
a. Eumycotic (fungal) mycetoma
 White grain: Pseudallescheria boydii, Acremonium falciforme
 Black grain: Madurella mycetomatis, Exophiala jeanselmei
b. Actinomycotic (bacterial) mycetoma
 Nocardia, Streptomyces, Actinomyces
2. Distribution
a. Most occur in tropical and subtropical regions of world
b. Infections in temperate regions do occur (including U.S.)
3. Clinical disease
a. Chronic granulomatous infection
b.
Triad of symptoms
 Swelling (tumefactions) of subcutaneous tissue causing tumor-like deformities
 Multiple sinus tracts (fistula) that tunnel through subcutaneous tissue to surface
 Drainage contains sulfur granules (vary in color, contain polysaccharide
material and mycelium)
c. Slowly progresses to involve bone, muscle, or other contiguous tissue
4. Eumycotic (Fungal) Mycetoma etiologic agents
a. Acremonium falciforme
 Colony morphology
o Growth at 1-3 weeks
o Colony is grayish brown becoming grayish-violet; texture is glabrous to lightly
downy, sometimes powdery
o Reverse: pale
 Microscopic examination
o Hyaline septate hyphae
o Phialides solitary long and narrow, typically with a septum at
the base and a scarcely visible collaret at the apex
o Conidia oblong to ovoid, unicellular, often accumulating in
clusters at the apices of the phialides
5. Actinomycotic (Bacterial) Mycetoma etiological agents
a. Nocardia species
 Colony morphology on routine agar (BAP, CHOC, SAB, BHI)
o Aerobic growth at 2-3 days
o Extremely variable
 Adherent
 Some isolates beta-hemolytic on BAP
 Wrinkled
 Often dry, chalky-white appearance to orange-tan pigment
 Crumbly
 Colony morphology on LJ agar
o Aerobic growth at 2-7 days
o Colony is orange and wrinkled
 Gram stain
o GPR – beaded, branching, fine, delicate filaments with fragmentation
 Partially acid fast positive
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 57
b. Streptomyces species
 Colony morphology on routine agar (BAP, CHOC, SAB, BHI)
o Aerobic growth at 3-10 days
o Variable
 Glabrous or waxy heaped colonies
 Morphology varies
 Gram stain
o GPR – extensive branching with chains and spores
o Does not fragment easily
 Acid fast negative
c.
Actinomyces species
 Colony morphology on AnaBAP
o Anaerobic growth at 3-10 days
o Most colonies are small, smooth, flat, convex, gray-white, and translucent
with entire margins
 A. israelii & A. gerencseriae – white, opaque, may resemble a “molar
tooth”
 A. odontolyticus – turns red after several days in ambient air and may be
beta-hemolytic
 Gram stain
o GPR – branching, beaded or banded, thin, filamentous
 Acid fast negative
C. Sporotrichosis
1. Etiologic agents
a. Sporothrix schenckii
2. Distribution
a. Worldwide
b. Found on living or dead vegetation
c. Farmers, nursery workers, gardeners, florists, miners
3. Clinical disease
a. Known as the “rose gardener’s” disease
b. Chronic subcutaneous infection
c. Primary lesion starts as a small, non-healing ulcer (on hand or finger)
d. With time, nodular lesions of skin or subcutaneous tissue at point of contact develop
e. Later, can involve lymphatic channels and lymph nodes draining the region
f. Only rarely, can the disease disseminate (systemic)
g. Also, a pulmonary infection may be seen
4. Sporothrix schenckii
a. Cultivation of mold phase
i. Media and growth requirements
 Sabouraud’s and Inhibitory Mold agars – good growth at 3-5 days
 Incubate at 25-30ºC in ambient air for up to 2-4 weeks
ii. Colony morphology
 Whitish to black colony on surface and reverse
 Texture glabrous, moist
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 58
iii. Microscopic morphology
 Hyaline, septate hyphae
 Conidiophores little differentiated from vegetative
hyphae
 Flowerette arrangement: conidia hyaline to brown,
ovoid, thin-walled, grouped in rosettes at the tip of
the conidiophore. Conidia are attached to
conidiophore by an individual, delicate, threadlike
structure (denticle)
 Sleeve arrangement: brown conidia, ovoid or
sometimes triangular, thick-walled, attached directly
to the sides of hyphae
 Can be confused with Acremonium (no yeast
phase)
b. Cultivation of yeast phase
i. Media and growth requirements
 Brain heart infusion agar supplemented with blood
 Incubate at 35ºC in ambient air for 1-5 days
i. Colony morphology
 Cream to beige colony with creamy texture
iii. Microscopic morphology
 Yeast ovoid, elongate or cigar-shaped, producing one or several buds
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi
Page 59
Subcutaneous Fungi Case Studies
Directions - Within CLS419 Clinical Microbiology II, go to Assignments, Clinical Rotation – Mycology,
and then Subcutaneous Fungi Unit. Go to the corresponding case study to view the microscopic
examples for each case. Click on an image to see an enlarged view. Answer the following short
answer or single answer multiple choice questions for each case.
Case 1
This organism was isolated from a 35-year-old female's arm lesion. The Gram stain is of growth
obtained at 35ºC. The wet preparations are of growth obtained at 25ºC.
1. Identify this fungus.
2. A common way humans become infected with this organism is by having contact with:
A. Cats and dogs
B. Pigeons
C. Poultry
D. Rose bushes
3. Clinically, this organism is most associated with a lesion:
A. Developing at the site of infection, spreading via the lymphatic system; may become systemic
B. Developing as a result of systemic spread following a respiratory infection
C. Remaining localized to the area of initial contact resulting in a single lesion
D. Remaining localized to tissue that contains keratin
Case 2
Shown are direct specimen stains of drainage from an edematous, foot wound from a 60-year-old
farmer.
1. Based on the clinical and laboratory information, what genus would you suspect?
A. Actinomyces sp.
B. Aspergillus sp.
C. Mycobacterium sp.
D. Nocardia sp.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi – Case Studies & Study Questions
Page 60
Subcutaneous Fungi – Study Questions
Matching:
1. Match each of the following types of sporulation with its description.
Sporulation type
____
Cladosporium
____
Phialophora
____
Acrotheca
Description
a. Production of flask-shaped or urn-shaped phialides
ranging from 4 to 7 um in length, terminating in a
collarette from the neck of which tight clusters of hyaline
conidia are formed.
b. Short chains of conidia arise from short denticles that are
attached laterally to the sides of conidiogenous cells,
which are borne sympodially in circumferential
arrangement.
c. Hyphae are freely branching, forming long chains of darkstaining, elliptical conidia. A scar (dysjunctor) is formed
where each conidium becomes detached
2. Match the fungi with their microscopic characteristics.
Fungi
____
Sporothrix schenckii
____
Acremonium species
Microscopic Characteristics
a. Mold phase has delicate hyphae producing fruiting
heads characterized by several tiny, elliptical conidia
arranged in a daisy petal fashion at the tip of a
delicate conidiophore. Yeast phase has elongated,
cigar-shaped blastoconidia.
b. Mold phase has septate hyphae with slender phialides
tapering at the end and giving rise to a cluster of
conidia. There is no yeast phase.
3. Match each of the following diseases with its description.
Disease
____
Chromoblastomycosis
____
Eumycotic mycetomas
____
Actinomycotic mycetomas
Description
a. Caused by true fungi, with symptoms including
swelling, sinus tracts and sulfur granules
b. Caused by true fungi with symptoms including
tumor-like lesions with sclerotic bodies found in the
tissues
c. Caused by a group of branching, gram-positive,
filamentous bacteria, with symptoms including
swelling, sinus tracts and sulfur granules
Multiple Choice:
4. Which of the following fungi are able to produce all three types of sporulation (cladosporium,
phialophora, and acrotheca)?
a. Phialophora
b. Cladophialophora
c. Fonsecaea
d. All of the above
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi – Case Studies & Study Questions
Page 61
5. Differentiate between the following fungal-like or branching bacteria including gram stain
morphology, growth requirements (media and atmosphere), and acid-fast reaction.
Bacteria
Gram
stain
Media of
choice
Atmospheric
requirements
Acid fast
reaction
Nocardia
species
Streptomyces
species
Actinomyces
species
Mycobacterium
species
CLS419 Clinical Microbiology II
Rotation II
Mycology
Subcutaneous Fungi – Case Studies & Study Questions
Page 62
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Seven:
Systemic Dimorphic Fungi
CLS419 Clinical Microbiology II
Rotation II
Mycology
Systemic Dimorphic Fungi
Page 63
SYSTEMIC DIMORPHIC FUNGI
Objectives:
Upon completion of this CLS 419 unit, the Clinical Laboratory Science student will:
1.
Explain the laboratory process of converting dimorphic fungi from mold to yeast.
2.
Correlate the clinical, epidemiological and laboratory findings, including macroscopic and
microscopic morphology at both room temperature and 370 F and growth rate, for the following
organisms:
a. Histoplasma capsulatum
b. Blastomyces dermatitidis
c. Coccidioides immitis
d. Sporothrix schenkii
3.
Differentiate Histoplasma capsulatum from the opportunist Sepedonium species.
4.
Discuss the use of molecular diagnostics in the identification of dimorphic fungi.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Systemic Dimorphic Fungi
Page 64
Systemic Dimorphic Fungi
I.
Basic Characteristics
A. Dimorphic – two morphological stages
1. Mycelial stage at 25-30ºC
2. Parasitic stage (yeast or spherule) in human host (35-37ºC)
3. Definitive diagnosis: must demonstrate both stages (prove dimorphism)
B. Epidemiology
1. Found primarily in North America (except Paracoccidioides is in South America)
2. Soil, decaying vegetation, bird and bat droppings
C. Clinical Significance
1. Man is incidental host
2. Pathogens
3. Acquire by inhalation and develop respiratory infection
4. Can then become systemic
a. Involvement of internal organs including lymph nodes, bone, subcutaneous tissue
and skin
b. May be asymptomatic
c. Immunocompromised and long-term corticosteroid therapy patients most prone
5. Diseases / Etiologic agents
a. Histoplasmosis / Histoplasma capsulatum
b. Blastomycosis / Blastomyces dermatitidis
c. Coccidioidomycosis / Coccidioides immitis
II. Histoplasmosis
A. Etiologic agent
1. Histoplasma capsulatum
a. Endemic in Missouri, Mississippi, and Ohio River deltas
B. Pathogenesis
1. 95% of cases are asymptomatic
2. Infection begins in lung and eventually invades the reticuloendothelial system
3. Can disseminate to lymphatic tissue, liver, spleen, kidneys, meninges, and heart (esp. in
immunocompromised patients)
C. Laboratory Diagnosis
1. Specimens
a. Sputum, blood, bone marrow
b. Urine, oral lesion scraping, lymph node biopsy, liver, etc.
2. Direct detection methods
a. Giemsa or Wright’s stains
i. Look for small, round to oval yeast cells within mononuclear cells (intracellular)
ii. Must distinguish from Leishmania and Toxoplasma
b. Calcofluor white stain
c. PAS, Methenamine silver stain, H&E stain on tissue sections
i. Observe for intracellular yeast cells
3. Cultivation of mold phase
a. Media and growth requirements
i. Sabouraud’s and Inhibitory Mold agars – good growth at 2-4 weeks
 If many organisms present, may see growth at soon as 3-5 days
ii. Mycosel agar – no growth
iii. Incubate at 22°C in ambient air for up to 4-6 weeks
CLS419 Clinical Microbiology II
Rotation II
Mycology
Systemic Dimorphic Fungi
Page 65
b. Colony morphology
i. White colony becoming brownish on the surface and yellowish on reverse
ii. Texture wooly to granular
c. Microscopic morphology
i. Hyaline, septate hyphae
ii. Macroconidia are unicellular, hyaline, thick-walled, smooth or warty (tuberculate)
iii. Microconidia are unicellular, hyaline, with wall smooth or rough
iv. This is the diagnostic form
v. Can be confused with Sepedonium species (does not have yeast phase)
4. Cultivation of yeast phase
a. Media and growth requirements
i. Blood enriched media such as BHI
ii. Incubate at 35°C in ambient air for 2-4 weeks
b. Colony morphology
i. Creamy texture with cream color on surface and reverse
c. Microscopic morphology
i. Small budding yeast measuring 2-4 um in length
5. Serological testing
a. Double immunodiffusion (exoantigen test)
b. Complement fixation
III. Blastomycosis
A. Etiologic agent
1. Blastomyces dermatitidis
a. Endemic in Mississippi and Ohio River basins
B. Pathogenesis
1. Causes an acute or chronic suppurative and granulomatous infection
2. Infection begins in lung by inhaling conidia or hyphal elements
3. Can spread and involve lungs, long bones, soft tissue and skin
C. Laboratory Diagnosis
1. Specimens
a. Cutaneous lesions
b. Sputum
c. Biopsy of affected organ
2. Direct detection methods
a. KOH and Calcofluor White preparation
i. Look for large, spherical, refractile yeast that have a double-contoured wall and
buds connected to the parent cell by a broad base
ii. Size is 8-15 um
3. Cultivation of mold phase
a. Media and growth requirements
i. Sabouraud’s and Inhibitory Mold agars – good growth at 1-4 weeks
ii. Mycosel agar – no growth
iii. Incubate at 22°C in ambient air for up to 4-6 weeks
b. Colony morphology
i. White, tan, or brown colony on surface, reverse is pale to brownish
ii. Texture cottony or glabrous
CLS419 Clinical Microbiology II
Rotation II
Mycology
Systemic Dimorphic Fungi
Page 66
c.
Microscopic morphology – nondescript
i. Hyaline, septate hyphae
ii. Conidiophores are short, unbranched
iii. Conidia are borne on short lateral branches that are ovoid to dumbbell shaped
4. Cultivation of yeast phase
a. Media and growth requirements
i. Cottonseed conversion agar
ii. Incubate at 35°C in ambient air for 1 week
b. Colony morphology
i. White to beige colony that is creamy, granular to verrucose in texture
c. Microscopic morphology
i. Yeast with refractile walls, budding on a broad base
ii. This is the diagnostic form
5. Other test methods
a. Serological testing
i. Double immunodiffusion (exoantigen test)
b. Nucleic acid amplification assay
i. Replacing serological testing
IV. Coccidioidomycosis (San Joaquin Valley Fever)
A. Etiologic agent
1. Coccidioides immitis
a. Endemic in San Joaquin Valley, CA; Maricopa and Pima counties of AZ, and
southwestern TX
B. Pathogenesis
1. 60% of infections are asymptomatic and have self-limited respiratory tract infections
2. Infection begins in lung by inhaling arthroconidia (very infectious – 10 conidia inhaled will
infect)
3. Can disseminate (1%) with extension to visceral organs, meninges, bone, skin, lymph
nodes, and subcutaneous tissue
C. Laboratory Diagnosis
1. Specimens
a. Sputum
b. Biopsy of affected organ
2. Direct detection methods
a. KOH preparation, Calcofluor white stain
i. Look for nonbudding, thick-walled spherule, 30-60 μm in diameter, containing
either granular material or numerous small nonbudding endospores
ii. Will not see a yeast form
b. PAS, Methenamine silver stain, H&E stain on tissue sections
3. Cultivation of mold phase
a. BIOHAZARD to lab workers, use strict safety precautions when examining cultures
(must do all work in biological safety hood, wear gown, gloves and mask, and seal all
culture plates)
b. Media and growth requirements
i. Sabouraud’s, Mycosel and Inhibitory Mold agars – good growth at 3 days to 3
weeks
ii. Blood agar – growth
iii. Incubate at 22°C in ambient air for up to 4 weeks
CLS419 Clinical Microbiology II
Rotation II
Mycology
Systemic Dimorphic Fungi
Page 67
c.
Colony morphology
i. Rapid growth
ii. White to gray colonies (sometimes beige, pink, cinnamon, yellow or brown) on
surface, reverse pale, sometimes orange, or pale to dark brown
ii. Texture wooly to glabrous
d. Microscopic morphology
i. Hyaline, septate hyphae
ii. Conidiophores absent
iii. Arthroconidia unicellular, rectangular to barrel shaped, often somewhat wider in
diameter than the hyphae, alternating with empty cells (disjunctors)
e. Mold phase will grow at both 22°C and 35°C
4. Cultivation of spherule phase
a. Not recommended
i. Use of special media incubated at 42ºC
ii. Experimentally infect lab animals and perform tissue biopsy
5. Other test methods
a. Serological testing
i. Double immunodiffusion (exoantigen test)
b. Nucleic acid amplification assay
CLS419 Clinical Microbiology II
Rotation II
Mycology
Systemic Dimorphic Fungi
Page 68
Systemic Dimorphic Fungi Case Studies
Directions - Within CLS419 Clinical Microbiology II, go to Assignments, Clinical Rotation – Mycology,
and then Systemic Dimorphic Fungi Unit. Go to the corresponding case study to view the microscopic
examples for each case. Click on an image to see an enlarged view. Answer the following short
answer or single answer multiple choice questions for each case.
Case 1
This fungus was isolated from a left, lower-lobe lung biopsy from a 17-year-old woman. The woman
started experiencing pulmonary symptoms in Chicago after living in Phoenix, Arizona for three years.
A mature colony was noted on day 4 of incubation at 25ºC.
1. Identify this fungus.
2. What structures would you expect the histologic stains of this lung biopsy to reveal?
A. Ascospores
B. Broad-based, budding yeast
C. Endosporulating spherule
D. Small, single budding yeast
Case 2
Shown is a Periodic-Acid-Schiff (PAS) stain of a lung biopsy from a 58-year-old male who complained
of a chronic, productive cough. He smoked two to three packs of cigarettes a day for the last 35-40
years. Upon x-ray, a homogeneous mass was noted in his left lung. The wet preparations are of the
organism growth after 12 days of 35ºC incubation.
1. Identify this fungus.
2. The microscopic morphology of this organism’s growth at 25C characteristically exhibits:
A. Alternating arthroconidia
B. Indistinctive septate hyphae with small, round or pear-shaped conidia on conidiophores
C. Rosette-like clusters of microconidia around tapered conidiophore
D. Tuberculate macroconidia w/clustered microconidia
Case 3
Shown is a Gomori-methenamine silver stain of caseous, lung lesion material from a 20-year-old
female. The wet preparations are from growth on Inhibitory mold agar after 16 days incubation at
25ºC.
1. Identify this fungus.
2. Clinically, this organism presents as a(an)
A. Lesion developing at the site of infection, spreading via the lymphatic system; may become
systemic
B. Localized cutaneous lesion that rarely spreads to the respiratory tract
C. Nonpathogenic contaminant
D. Respiratory infection that may exhibit systemic spread
CLS419 Clinical Microbiology II
Mycology
Rotation II
Systemic Dimorphic Fungi – Case Studies & Study Questions
Page 69
Systemic Dimorphic Fungi – Study Questions
Five fungal species share the common characteristics of growing in a mold form at ambient (room)
temperature and in a yeast form when incubated at 35°C. These “dimorphic” fungi also are obligate
pathogens for humans with the potential for causing disseminated, deep-seated infections. They
generally are slow growing when recovered from clinical specimens and can be identified either by
recognizing the microscopic morphology of the mold form when recovered in cultures or by detecting
the characteristic yeast or spherule forms in stained tissue sections.
1. Give a brief description of the microscopic features of both the mold and yeast forms of the
following species of dimorphic fungi:
Dimorphic fungi
Mold form
Yeast form
Blastomyces
dermatitidis
Histoplasma
capsulatum
Coccidioides
immitis
2. What culture criteria are used to determine if an unknown isolate might be suspected of being a
dimorphic mold?
a. Slow grower (2-4 weeks)
b. Grows as mold at 22°C and converts to yeast form at 35°C
c. Grows on media containing antifungal agents
d. All of the above
e. A and B only
3. For Histoplasmosis, Coccidioides and Blastomyces infections, the common body site where
infection begins is:
a. Bladder
b. Brain
c. Lungs
d. Liver
4. Discuss three safety precautions that should be met when working with the mold forms of
systemic pathogens.
5. Compare and contrast the colony and microscopic characteristics of Histoplasmosis capsulatum
and Sepedonium species
CLS419 Clinical Microbiology II
Mycology
Rotation II
Systemic Dimorphic Fungi – Case Studies & Study Questions
Page 70
CLS419 – Clinical Microbiology II
Mycology Manual
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Eight:
Appendix
CLS419 Clinical Microbiology II
Rotation II
Mycology
Appendix
Page 71
Mycology Procedures
10% KOH Preparation
1. Add a drop of 10% KOH to specimen on slide. Coverslip.
 Gentle heating may aid in dissolving debris
 If specimen is thick, it may take 15-30 minutes to dissolve
2. Observe under low light or with phase scope
Calcofluor White Stain
1. Add drop of Calcofluor White stain to specimen on slide. Coverslip.
2. Allow to sit approximately 3 minutes.
3. Use a fluorescent scope and look for apple green fluorescence.
Cornmeal Tween 80 Morphology
1. With loop, make one streak into the agar down the center of an
area and 3 or 4 parallel cuts across the first ½ inch or 1 cm apart,
holding the inoculating wire at about a 45º angle to dilute inoculum.
2. Incubate 24-72 hours at 30ºC
3. Place coverslip on surface of the agar, covering inoculation streaks
4. Remove petri dish lid and examine through the coverslip with the
microscope using the 10x and 40x objectives. Look for the most
characteristic morphology near the edge of the coverslip.
Germ Tube
1. Emulsify small amount of yeast in tube containing 0.5-1.0 ml serum (rabbit, fetal calf, or
human)
2. Incubate at 35ºC for 3 hours
3. Place one drop of suspension on a slide, coverslip, and examine microscopically (40x) for
long tube-like projections (germ tubes) extending out form the yeast cells.
4. Do not confuse germ tube with pseudohyphae (constricted at point of attachment)
Pseudohyphae
Germ Tube
 Parallel sides
 Not necessarily parallel
 Non-septate
 May be septate
 No constriction at point of attachment
 Constricted at point of attachment
(sausage shaped)
India Ink Preparation
1. Make a .5 McFarland suspension of yeast in sterile saline
2. Place one drop of suspension with one drop of India ink (be careful with ink) on microscope
slide. (Strive for a thin smear)
3. Coverslip (drain on paper towels if coverslip is “floating”).
4. Let sit (up to 10 minutes) to allow cells to settle
5. Observe microscopically using 10x and 40x objective with condenser adjusted for maximum
light. Look for a clear capsule around yeast.
CLS419 Clinical Microbiology II
Rotation II
Mycology
Appendix
Page 72
Urease
1. Streak slant using sterile loop (use LOTS of organism)
2. Incubate at 35ºC
3. Observe at 3 hours and at 24 hours.
Scotch Tape Preparation
1. Lightly press transparent scotch tape to colony
2. Affix tape to slide that has a drop of lactophenol cotton blue stain on it
3. Observe under light microscope at 10x and 40x
Slide Culture
1. Setup equipment and supplies: Petri dish, paper towel, sterile water, two sticks, two
microscope slides, coverslips.
 Place slide on 2 sticks in Petri dish (gauze or paper towel under sticks moistened with
sterile water)
2. Using sterile scalpel, cut square of Sab-dex agar or Potato Dextrose agar smaller than
coverslip and place on slide. Two pieces of agar can be placed on the slide to provide
duplicate cultures.
3. Inoculate all four SIDES of agar square with mold (use dissecting needles to pick up mold)
4. Place a coverslip on top of each agar square
5. Make sure paper towel (or filter paper) is WET with excess water in bottom of Petri dish
6. Tape plate and incubate at room temperature 3-5 days.
7. When good amount of growth is evident, remove coverslip and place on slide containing a
drop of lactophenol cotton blue stain
 Do just one coverslip in case mold is not mature enough
 Coverslip can be sealed with fingernail polish
8. Observe for characteristic fungal structures under light microscope using 10x and 40x
objectives.
Tease Preparation
1. Transfer piece of colony to slide and tease apart with dissecting needles
2. Add lactophenol cotton blue stain and coverslip
3. Observe under light microscope at 10x and 40x
CLS419 Clinical Microbiology II
Rotation II
Mycology
Appendix
Page 73
University of Nebraska Medical Center
Clinical Laboratory Science Program
CLS419 – Microbiology II
Mycology Lab Worksheet
Please document the Gram stain smears, LPAB preps, and other slides that you evaluate during your
Lab Activities each day. This worksheet is for your use to keep track of what you need to look at
each day and to have a record of what you saw to help you study. You may want to include drawings
of fungi and any other information that is helpful to you.
You will be tested over your objectives (these organisms are NOT the only ones on the exam).
Slide boxes may differ in organisms, you may not have all slides present in your box or you may have
additional organisms/slides to add to your list. This exercise is to gain experience with microscopy
work in mycology.
Slide ID
Fungus seen
(and other information)
Colony morphology
on SAB agar?
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
CLS419 Clinical Microbiology II
Rotation II
Mycology
Appendix
Page 74
Slide ID
Fungus seen
(and other information)
Colony morphology
on SAB agar?
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
Stain Type:
Organism:
CLS419 Clinical Microbiology II
Rotation II
Mycology
Appendix
Page 75
Mycology Tutor, Table of Contents
Introduction
Overview
Mycology
Taxonomy
Safety
Fungal Detection
Direct Techniques
India Ink
Microscopic
Procedure – 3 steps
(videos)
KOH
Microscopic
Procedure – 2 steps
(videos)
KOH/Calcofluor
Gram Stain
Gram-Weigert Stain
Histologic Stains
Isolation Techniques
Isolation media
Selective media
Organism Identification
Yeasts
Macroscopic
Background
Color
Shape
Texture
Differential Media
Microscopic
Wet Mount
Microscopic
Mycology Tutor, University of Washington
Procedure – 4 steps
(videos)
Germ Tube
Microscopic
Procedure
Germ tube formation
(2 videos)
Corn Meal Agar
Microscopic
Procedure – 2 steps
(videos)
Ascospore Stain
Biochemicals
Assimilation tests
Automated instruments
Enzymatic testing
Further testing
Molds
Macroscopic
Background
Growth rates (video)
Color
Texture
Differential tests
Microscopic
Background
Stains
Tease preparation
Procedure – 4 steps
(videos)
Scotch tape preparation
Procedure – 5 steps
(videos)
Slide Culture
Procedure – 5 steps
(videos)
Hair perforation
Conidiogenesis
Background
Blastoconidia (video)
Annelloconidia (video)
Phialoconidia (video)
Poroconidia (video)
Aleurioconidia (video)
Chlamydoconidia
(video)
Sporangiospores
(video)
Disease Associations
Superficial Mycoses
Tinea versicolor
Clinical presentation
Direct exam
Malassezia furfur
Macroscopic
Microscopic (video)
Tinea nigra
Clinical presentation
Phaeoannellomyces
werneckii
Macroscopic
Microscopic
White piedra
Clinical presentation
Trichosporon beigelii
Macroscopic
Microscopic
Dermatophytoses
Clinical presentation
Direct examination
Epidermophyton
floccosum
Macroscopic
Mycology Appendix
Microscopic
Microsporum canis
Macroscopic
Microscopic
Trichophyton
mentagrophytes
Macroscopic
Microscopic
Trichophyton rubrum
Macroscopic
Microscopic
Trichophyton tonsurans
Macroscopic
Microscopic
Subcutaneous Mycoses
Sporotrichosis
Clinical presentation
Sporothrix schenckii
Macroscopic: room
temp
Microscopic: mold
phase (video)
Microscopic: yeast
phase
Chromoblastomycosis
Clinical presentation
Direct examination
Cladophialophora
carrionii
Macroscopic
Microscopic
Fonsecaea species
Macroscopic
Microscopic (video)
Phialophora verrucosa
Macroscopic
Microscopic (video)
Page 76
Mycetomas
Clinical presentation
Pseudallescheria boydii
Macroscopic
Sexual stage
Asexual stage
Exophiala jeanselmei
Macroscopic
Microscopic (video)
Filamentous bacteria
Phaeohyphomycosis
Clinical presentation
Wangiella dermatidis
Macroscopic
Microscopic
Cladophialophora
bantiana
Macroscopic
Microscopic
Exophiala spp.
Macroscopic
Microscopic
Phialophora richardsiae
Macroscopic
Microscopic
Systemic Mycoses
Cryptococcosis
Clinical presentation
Direct examination
Cryptococcus
neoformans
Macroscopic
Microscopic
Blastomycosis
Clinical presentation
Direct examination
Blastomyces
dermatitidis
Mycology Tutor, University of Washington
Macroscopic: room
temp
Microscopic: room
temp
Microscopic: 37
degrees
Coccidioidomycosis
Clinical presentation
Direct examination
Coccidioides immitis
Macroscopic: room
temp
Microscopic
Histoplasmosis
Clinical presentation
Histoplasma
capsulatum
Macroscopic: room
temp
Microscopic: room
temp (video)
Microscopic: 37
degrees
Paracoccidioidomycosis
Clinical presentation
Paracoccidioides
braziliensis
Macroscopic: room
temp
Microscopic: room
temp
Microscopic: yeast
phase (video)
Opportunistic Fungi
Overview
Common Opportunists
Candida albicans
Macroscopic
Direct examination
Microscopic
Aspergillus fumigatus
Macroscopic
Microscopic
Animation (video)
Aspergillus flavus
Macroscopic
Microscopic
Animation (video)
Rhizopus species
Macroscopic
Microscopic
Mucor species
Macroscopic
Microscopic
Pneumocystis carinii
Less Common Opportunists
Acremonium species
Macroscopic
Microscopic
Animation (video)
Alternaria species
Macroscopic
Microscopic
Animation (video)
Chrysosporium species
Macroscopic
Microscopic
Chaetomium species
Macroscopic
Microscopic
Curvularia species
Macroscopic
Microscopic
Animation (video)
Epicoccum species
Macroscopic
Microscopic
Mycology Appendix
Fusarium species
Macroscopic
Microscopic
Paecilomyces species
Macroscopic
Microscopic
Animation (video)
Penicillium species
Macroscopic
Microscopic
Animation (video)
Phoma species
Macroscopic
Microscopic
Pithomyces species
Macroscopic
Microscopic
Scopulariopsis species
Macroscopic
Microscopic
Ulocladium species
Macroscopic
Microscopic
Animation (video)
Exams
Take Exam 1
Page 77