Mycology 2016

1. MYCOLOGY
Margie Morgan, PhD D(ABMM)
Just the basics – meant for board
review or brief study of this
fascinating area of microbiology!
Mycology

2. Starting point
Yeast are:
• Unicellular / produce budding daughter cells
• Colony on solid media are usually white to beige and
appear much like bacterial colonies
• Some genera produce mucoid colonies
(Cryptococcus)

3. Starting point
Molds are:
• Filamentous with hyphae
• Produce conidia [spores]
• Colonies on solid agar are downy, fluffy, cottony
• Most mold colonies are pigmented which aid in
identification
hyphae
spores

4. Appropriate Specimens and Transport
conditions for Fungal disease diagnosis
• Fungi are very hardy organisms
• No requirement for special transport media for culture
submission
• Sterile containers used to prevent bacterial contamination
• Numerous sites are appropriate for culture
• Respiratory specimens – sputum, bronchial lavage,
brushings, nasal sinuses
• Tissue biopsies
• Cutaneous - Skin scrapings, material from lesions
• Ocular
• Sterile body fluids including CSF
• Blood, bone marrow

5. Fungal Culture Media
• Sabouraud’s glucose agar (SABS)

6. Common Fungal Media
• Mycosel/Mycobiotic agars
• Selective SABS with chloramphenicol and cycloheximide
• Used for culture of dermatophytes – fungi that cause skin, hair and nail
infections
• Brain heart infusion agar
• Primary recovery of all fungal organisms
• Can make it more selective by adding chloramphenicol and
cycloheximide
• Can add blood to agar to nurture systemic fungi
• Fungal cultures incubated for 4 weeks at 30˚C , room air
• Lower temperature than bacterial culture incubation [35˚C]
• If plates are used for fungal cultures the plates must be sealed with
air permeable tape for laboratory safety

7. What effect does Cycloheximide have
when added to media?
• Prevents rapidly growing molds from overgrowing
dimorphics and dermatophytes
• This is the positive aspect of cycloheximide in media
• Beware: it is not all good, it can suppress important
fungi from growing. Inhibited fungi include:
• Trichosporon beigelii
• Candida tropicalis
• Cryptococcus neoformans
• Yeast phase of Blastomyces
• Yeast phase of Histoplasma

8. Inoculate fungal media
Seal plates with tape to prevent culture
contamination and escape of fungal spores
Incubate at 30˚C for 4 wk
If growth occurs - perform proper identification methods:
Yeast identification methods
Manual and automated biochemical reactions capable
of identifying most yeast species.
Newer methods [Mass spectrometry – MALDI-TOF and
16 sRNA sequencing
Processing of Fungal Cultures - stepwise

9. Lactophenol cotton blue [LCB] adhesive tape preparation is
method used for mold identification.
LCB mounting medium consists of phenol
lactic acid, glycerol and aniline cotton blue dye.
Clear adhesive (scotch) tape touches a mold colony, picking
up fungal hyphae and pressed into one drop of LCB
on a microscope slide.
If LCB prep is not able to
identify a mold 16sRNA
sequencing can be used
to identify problematic
molds in reference
laboratories.
Mold Identification methods

10. Safety in the Mycology Laboratory
• If a culture is growing a mold, it cannot be opened on the
bench top
• All mold work must be performed in a BSL-2 biosafety
cabinet with Hepa filtration
• Yeast identification can be performed on the bench top

11. Direct Exams used to identify fungi
directly from patient specimens
• Gram stain – all specimen types can be Gram stained.
Can only reliably detect yeast by Gram stain.
• KOH preparation – Skin, Hair or Nails examined for
both yeast and/or hyphae
• Calcofluor white stain – all specimen types can be
stained and examined for yeast and/or hyphae
• India ink – Primarily used for CSF for the detection of
Cryptococcus neoformans and C. gattii

12. Yeast cells stain blue [Gram positive]. Examine for
budding cells to confirm that it is a yeast cell and not
an artifact. Examination on oil immersion lens.
You can also detect pseudohyphae on Gram stain.
Mold can be difficult to identify on a Gram stain.
pseudohyphae
mold
pseudohyphae
Gram Stain

13. Used to detect yeast and/or hyphae in skin, hair
and nail specimens using 40X light microscope.
KOH dissolves keratin found in cell material
and frees hyphae from the cell
KOH exams can be difficult to interpret!
KOH – potassium hydroxide prep

14. Yeast, pseudohyphae, and mycelial fungi bind with
the Calcofluor white stain.
Prep is interpreted using a fluorescence microscope.
Sensitivity and specificity is improved over the KOH
preparation.
Calcofluor white stain

15. One drop of black ink is
placed into one drop of CSF
and examined using a 40X
lens on light microscope
It is a “negative” stain,
staining the background not
the yeast cell
The clearing is the
polysaccharide capsule of
Cryptococcus neoformans or
C. gattii. Specificity is
improved if you look for
budding yeast cells.
India Ink

16. Methenamine Silver Stain [GMS] –
yeast and hyphae stain grey to black.
Examine the hyphae for presence of septations in the hyphae,
broad or more narrow width and angle of branching.
Examine the size and budding pattern of observed yeast.
We will observe on later slides how these criteria can assist in
identification.
Examination of fungi in fixed tissue

17. PAS-positive staining red against a green or
blue background
Periodic Acid Schiff [PAS]

18. Stain-Cryptococcus neoformans polysaccharide capsule
stains pink
Mucicarmine [Mucin] stain

19. Great for cellularity, but
GMS, or PAS better show
features of the fungi.
Hematoxylin and Eosin Stain

20. DIMORPHIC FUNGI
Important systemic pathogens with some
unique characteristics

21. What does Dimorphic mean?
• Two forms exist for one fungus species depending on
temperature and conditions of environment
• Mycelial form - Hyphae and conidia
• free living form found in nature and at laboratory temperature <=30˚C
• Yeast or yeast like form
• parasitic phase found in human tissue or in the lab >= 35˚
Histoplasma capsulatum – mold
from 30˚C culture
Histoplasma capsulatum – yeast
from tissue and 35˚C culture

22. Dimorphic Fungi capable of causing
systemic infection – most common
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis
• Paracoccidioides brasiliensis
• Sporothrix schenckii
• Penicillium marneffei

23. Histoplasma capsulatum
• World wide distribution / In USA in Ohio, Missouri,
and Mississippi River valleys considered endemic
• Associate with Bat guano (Spelunker = cave
explorers) and bird droppings

24. Histoplasmosis Disease
• 95% of infections are subclinical
• 5% infections:
• Progressive pulmonary
• Chronic systemic infection with dissemination to the RES
system including bone marrow
• Acute fulminating systemic disease (fatal)
• Reactivation disease can occur in elderly and
immunosuppressed (AIDS is a good example)
• Bone marrow exam is useful in diagnosing disseminated
infections
• Mucocutaneous lesions are a unique &
common site of dissemination

25. The yeast of Histoplasma capsulatum
prefer to be intracellular and inhabit
Macrophages.
Small 2 – 4 um, regular in size, and oval to round.
Do not have a capsule, this is just a staining artifact.

26. H & E PAS
Gram Wright’s
Histoplasma will stain with a variety of stains

27. Histoplasmosis rapid
diagnosis
• Antigen detection in urine
• Quantitative Enzyme immunoassay
• Random urine specimen
• Most useful for disseminated infection and chronic
pulmonary disease
• Antigen is detectable in >=85% of these infections
• Good for diagnosis of immune suppressed patients
that do not produce a detectable antibody response
• Antibody tests are available but have been replaced
mostly by Antigen detection

28. Histoplasma
capsulatum
• Fungal culture incubated at 30˚C
• Very SLOW growing taking 2 – 8 weeks to form colonies
• Colony is white to brown and cottony
• Microscopic appearance – tuberculated macroconidia that are
large and round (8 – 16 µM) plus small microconidia (2 - 4µM)
[see picture]
• Microconidia are the infectious particle growing in nature and
capable of penetrating deep into the lung
• DNA probe must be used to confirm identification so there is no
confusion with look alike fungi to the inexperience
• Sepedonium species looks somewhat like Histoplasma and is
considered a look a like fungus – it is a contaminate

29. Histoplasma capsulatum
culture at 30˚C is white and
cottony.
Microscopic exam:
Tuberculate [projections]
on the macroconidia.
Microconidia are the infectious
particle. Thin hyaline septate hyphae
Appearance in culture at 30 degrees C
Sepedonium
species

30. Appearance in culture at
35 degrees C
• Culture at 35˚C is yeast phase
• Grow as small yeast, round to oval, always consistent
in size and shape (2 -4 uM) narrow neck at the budding
juncture

31. Histoplasma capsulatum in tissue
• Granulomas are usually produced and can be either
caseating or non caseating
• Infection usually begins by breathing in the microconidia
and infecting the Lung
• Infection disseminates to organs of the
Reticuloendothelial System (RES) – with high % of
dissemination to the Bone Marrow
• Intracellular budding yeast (2 – 4 µM) are seen in all
tissues

32. Leishmania species
Note small round
kinetoplast next to nucleus
Toxoplasma
Histoplasma capsulatum
Beware of look alike organisms
in tissue specimens!!

33. H capsulatum var duboisii yeast cells are 8 –
10 uM in size, which is 2X the size of regular
Histoplasma capsulatum yeast cells.
H. capsulatum var duboisii disease
is found in Central Africa
Causes infection in skin and bone
The 30˚C culture is identical to
H capsulatum.
Unusual variant of H. capsulatum

34. Blastomyces dermatitidis
• Epidemiology
• Ohio and Mississippi River valleys
• No association with specific animal or activity
• Forrest and river banks?
• Primarily a pulmonary infection which may disseminate to the skin
and bone
Well demarcated skin lesion is typical
Scraping of skin lesions are full of
yeast cells

35. Blastomyces dermatitidis
• Culture at 30˚C
• Grows in 2- 3 weeks
• Fluffy white – buff colored mold, prickly
• Pear shaped conidia at the end of supporting hyphae
– looks like lollipops
• Look alike fungus – Chrysosporium species
• Do DNA probe test to confirm identification
Blastomyces
Chrysosporium

36. Slow growing yeast colony taking @ 4
weeks to form a colony
Yeast cell is 8 – 20 um in size and is unique
for it’s Broad Based Budding pattern and the
double contoured wall.
Blastomyces culture at 37 degrees
C

37. Blastomyces dermatitidis histopathology
Mixed pyogenic and granulomatous inflammation is
observed in tissue with
Broad based budding yeast cells

38. Coccidioides immitis
• Endemic in SW USA, Mexico,
South America, in areas known
as the Sonoran life zone with a warm climate
and desert sands
Infection is from inhalation of fungal particles
found in the sand
• Coccidioides posadasii is a genetically related
to C. immitis. The two species are located in
different endemic regions, but produce the
same disease process

39. Coccidioidomycosis
• 95% of infections are asymptomatic or with limited
symptoms
• The remaining 5% are focal pulmonary, progressive
pulmonary or disseminated infections. Dissemination to
the central nervous system is difficult to cure and has a
high fatality rate.
• Higher incidence of dissemination occurs in patients
with:
• defects in cell mediated immunity (HIV),
• darker skinned ethnic groups,
• pregnancy

40. Coccidioides immitis [posadasii]
• Culture at 30˚C
• Requires only 2 – 3 days to grow, colony starts waxy
and becomes wooly in around 7 – 10 days
• Under the microscope one looks for foci of septated
hyphae with thick walled barrel shaped arthroconidia
with clear spaces in between. The clear spaces are
dead arthroconidia.
• Arthroconidia infectious particle in nature
• Very infectious to laboratory personnel

41. Coccidioides• Malbranchea species can look like C. immitis under the
microscope
• Because of look-a-like fungi one needs to confirm
identification of Coccidioides immitis with DNA probe or
similar method to be sure!
Coccidioides Malbranchea

42. Barrel shaped alternating arthroconidia are produced
in cultures grown at both 30 and 35 C.
There is no yeast phase for C. immitis [posadasii]
No yeast phase with Coccidioides!

43. Coccidioides Histopathology
• Thick walled spherules (10 – 80 uM) with endospores
are seen in tissue. This is the second form of Cocci. No
yeast cells are produced in tissue for this fungus.
• Spherules are at all stages of development- fragmented
spherules to well formed with endospores
• Granulomatous inflammation with caseation is usually
observed

44. Development of Cocci spherules from the
inhalation of Arthroconidia from nature

45. Rhinosporidium seeberi (aquatic parasite) forms spherules but
much larger than the Cocci spherules - they are usually > 80
uM in size. Also R. seeberi almost always cause oral or nasal
mass lesions
Oral or nasal mass lesions
of Rhinosporidium seeberi
Coccidioides
spherules
Coccidioides is not the only spherule
forming organism!

46. Paracoccidioides brasiliensis
• South American Blastomycosis –
endemic area Brazil, Venezuela, Columbia
• Inhale infectious particle from soil
• >95% of infections in males, possibly due to
estrogen inhibition of mycelial to yeast
transformation
• Disease presentation:
1. Pneumonia
2. Disseminated infection to 2 or more organs
3. Extrapulmonary lesions on the face and oral
mucosa

47. Paracoccidioides
• Cultures at 30*C for mycelial phase are usually not
done due to slow growth and nonspecific sporulation
• Culture @ 37˚C
• Slow growing yeast
• Large yeast (10 – 30uM) with multiple daughter buds (2 – 10
uM) in size
• Unique yeast cell known as the Mariner’s wheel or Pilot’s wheel
yeast

48. Tissue Exam of
Paracoccidioides brasiliensis
If more than 2 buds off mother cell –
High likelihood it is Paracoccidioides
Granulomatous inflammation with Giant
Cells

49. Sporotrichosis
• Sporothrix schenckii
• Cutaneous inoculation of fungus from penetrating injury with a
spore or thorn (rose bush)
• Initial skin lesion w/wo ulceration
• Lymph-cutaneous spread – bone – systemic
• Pulmonary and CNS infections are rare but reported

50. Starts as one ulcerative lesion and then chains
Up the lymphatics – can involve lymph nodes
and bone

51. Sporothrix
schenckii
•MOLD PHASE
• 30*˚C growth in 3 -5 days
• Turns brown to black over time
• Septate hyphae with conidia in daisy wheel pattern
•YEAST PHASE
• At 37˚C small oval yeast cells,
elongated 2 – 5 µM, described as cigar bodies

52. Sporothrix schenckii
• Histology –
• Pyogenic – to – granulomatous inflammation
• Hard to find yeast in human tissue
• Asteroid body known as Splendore-Hoeppli phenomenon
can be seen, but not specific to Sporothrix, also seen in:
• Zygomycetes (Mucorales)
• Aspergillus
• Blastomycosis
• Candida

53. Daisy like spore arrangement
Sporothrix
schenckii

54. Green colony with red diffusable pigment
Uncommon dimorphic fungus
The only species of Penicillium that is dimorphic
Causes skin lesions in tropics and
Pneumonia in immune suppressed
Penicillium marneffei

55. Penicillium marneffei yeast like cells in tissue

56. SUBCUTANEOUS FUNGAL
INFECTIONS
Very unique structures in tissue!

57. Subcutaneous Fungal Infections
Most common will be described
• Mycetoma [2 types]
• Actinomycotic – caused by higher bacteria
• Eumycotic – caused by dark pigmented molds
• Chromomycosis [Chromoblastomycosis]
• Phaeohyphomycosis
• Sporotrichosis

58. Mycetoma
• First observed in India and known
as Madura Foot or Maduromycosis
• Found in the hot temperate parts of
the world
• Three criteria describe Mycetoma:
1. Lesions lead to swollen extremities
2. Draining sinuses
3. Sulfur granules observed in tissue and found
in the weeping drainage
• Fungus grows on organic debris in soil
• Implanted into subcutaneous tissue from trauma

59. Swollen extremity and draining sinus with
sulfur granules
Sulfur Granule
Mycetoma

60. Mycetoma
• There are two types of Mycetoma:
1. Actinomycotic mycetoma – caused by higher bacteria
species
2. Eumycotic mycetoma – caused by the black molds
• Actinomycotic Mycetoma
• 98% of cases of Mycetoma
• Nocardia species most common cause
• Sulfur granules formed in tissue and the granules vary in
color and contain a matrix of the filamentous bacteria

61. Gram stain as filamentous Gram positive bacilli – can be
poorly staining and appear speckled.
Nocardia are positive [red] on the Modified Kinyoun stain.
Modified acid-fast stain
[modified Kinyoun stain]
Gram stain of sputum
containing Nocardia
Nocardia

62. Edge of granule has thin filamentous bacteria
for both bacteria – Nocardia is modified acid fast
[PAF] positive and is aerobic bacteria. Actinomyces
is PAF negative and grows anaerobically.
Beware! Sulfur granule
caused by Actinomyces
israelii looks identical.
Actinomycotic sulfur granule -
Nocardia

63. Requires 3 – 5 days to
grow on agar media [Sabs,
5% Sheep’s blood agar
Colony is dry and crumbly
Musty smell
Total of 85 species
Nocardia asteroides is the
most common species
isolated from human
infection
Identification by HPLC
or molecular methods
Nocardia species cause mycetoma, and
can also cause Pulmonary and Brain infections

64. Eumycotic mycetoma – subcutaneous
infection caused by the black molds
Numerous species of pigmented/black fungi found
naturally in the soil can cause this type of infection
Cause @ 2% of cases of mycetoma
Traumatic implantation injects the mold into the
subcutaneous tissue
Most common species of black mold include:
Cladophialophora (Cladosporium) carrionii
Cladophialophora bantiana
Phialophora verrucosa
Fonsecaea pedrosoi
Exophiala species
Wangiella species

65. Eumycotic sulfur granule – the granule is
full of a matrix of thick fungal hyphae

66. Chromomycosis/Chromoblastomycos
is
• Three characteristics describe Chromomycosis
• Wart like lesions in subcutaneous tissue
• Sclerotic bodies observed in tissue
• Growth of dark/pigmented fungi
• Black mold naturally found in the soil cause
infection through abrasion/ implantation
• Black molds that can cause Chromomycosis:
• Cladophialophora [Cladosporium] carrionii
• C. bantiana
• Phialophora verrucosa
• Fonsecaea pedrosoi
• Exophiala species
• Wangiella species

67. Chromomycosis/Chromoblastomycosis
Wart like/Verrucous lesions
In subcutaneous tissue
Sclerotic Body/Medlar Body/
Copper Penny is the unique
structure found in tissue

68. Prototheca wickerhamii – the cause of
Protothecosis
• Algae without chlorophyll
• Causes skin lesions & nodules
• Most common in patients with suppressed immune
system
• Compare morula of Protothecosis to sclerotic body of
Chromomycoses

69. Phaeohyphomycosis
This infection is caused by traumatic implantation of dark
fungi into subcutaneous tissue
• Variety of infections but nodules/lesions most common
with/without dissemination
• Dark hyphae only observed in tissue

70. Black molds/Dark molds
also known as Dematiaceous
fungi
Black colored colonies and the
reverse [back of colony] is also
black
Naturally brown hyphae and
spores
One of the major causes of mold
growth due to water damage!

71. Black Molds – Dematiaceous fungi
• Black colonies
• Brown hyphae and spores
• Numerous species
• Difficult to identify
• All have one of four types of sporulation
• Rhinocladiella-like
• Cladosporium-like
• Phialophora-like
• Acrotheca-like

72. Rhinocladiella type
sporulation

73. Phialophora type sporulation

74. Cladophialophora
[Cladosporium type sporulation]

75. Acrotheca type sporulation

76. Exophiala species
Black Molds that can cause
Mycetoma/Chromomycosis/Phaeohyphomycosis
These are difficult to identify but viewing is necessary!
Cladophialophora bantiana

77. Wangiella dermatitidis
Phialophora verrucosa

78. Alternaria
Other black molds of
importance:

79. Bipolaris
australiensis
Very invasive
fungal infection:
Skin, nasal
sinuses, bone
brain

80. Curvularia lunata
Center cell is the
largest
Opportunistic fungal pathogen
Reported in patients with Bone Marrow
Transplantation and Aplastic anemia
Can cause unusual erythematous skin lesions

81. Exserohilum rostrum
• Associated with compounded pharmaceutical [steroid]
products contaminated with dust/dirt
• Used for infections into lumbar spine and knee joints for
pain management
• Meningitis
• Spinal abscess
• Synovial infections

82. Scedosporium apiospermum/
Pseudallescheria boydii
Cat fur-like colony

83. IMPORTANT YEAST
CAUSING HUMAN
INFECTION
Candida species
Cryptococcus neoformans &
Cryptococcus gattii

84. Cutaneous and Superficial Mycoses
• Candida species (@ 10 found in humans)
• Opportunistic pathogen involving skin or mucous
membranes from excessive exposure to moisture,
antibiotics, or immune suppression
• Yeast is from endogenous source – found as normal
flora in the GI and GU tracts and skin
• Variety of infections including: Thrush, vaginitis, skin
lesions, nail, diaper rash, to more serious infections like
fungemia and endoarditis.

85. Candida species
• Candida albicans – most common species causing @
60% of human yeast infections
• Candida glabrata, C. krusei, and C. tropicalis are
becoming more common in infection
• These 3 species are more likelyto be resistant to
Fluconazole
• Candida parapsilosis has emerged as a pathogen of
children and IV lines

86. Candida species
• Grow in 24 – 48 hours
• SABS, IMA, BAP
• Bacteria-like colony – pasty white
• Budding yeast – oval @ 7-8 um in size
form pseudohyphae (Yeast do not detach and form
elongated hyhae) Can appear like sausage links
• Exception **Candida glabrata is @ 4 µM in size and does
NOT form any pseudohyphae

87. Candida albicans
Identification
• Germ tube formation
• Incubate small amount of yeast in serum for 3-4hr at 35 ˚C
• Do not incubate >4 hr – this can lead to a false positive
reaction with C. tropicalis
• C. dubliniensis also positive (uncommon yeast isolate)
• Chlamydospore formation
• Growth on cornmeal agar >48 hrs
• Rudimentary structures
C. albicans
chlamydospore
C. glabrata only forms yeast
No pseudohyphae

88. ChromAgar for the
identification of Candida
Chromogenic substrates
Turn different colors with
4 different yeast species
Yeast with pseudohyphae

89. Candida Histopathology
• Pyogenic to granulomatous
• Usually observe yeast cells, pseudohyphae and/or hyphae
appearing structures
• Candida glabrata = smaller yeast cells and no pseudohyphae
GMS stain of Candida glabrataCandida species not glabrata

90. Cryptococcus
neoformans
• In nature forms a 2um non-encapsulated yeast cell. It is
associated with bird droppings (esp. pigeon). C
neoformans is enriched by the nitrogen in the droppings.
• Yeast cells are inhaled – travels through the pulmonary
system with hematogenous spread to brain and meninges
• Has tropism to the meninges
• Infects mostly compromised hosts - AIDS

91. Cryptococcus neoformans
• Irregular sized (2 – 20uM) yeast cells
• Polysaccharide capsule is virulence factor and it’s presence is used in
diagnostic tests for C. neoformans
• India ink exam of CSF is a negative staining method/capsule not
stained,
• Sensitive test for AIDS patients (90% sensitive)
• Cryptococcal antigen test – capsular polysaccharide is detected
in both CSF and serum,
• Test for diagnosis and can also follow recovery with falling titer /more
sensitive than India ink
• Grows on mycologic agars but is sensitive to cycloheximide –
• Mucoid colonies due to capsule polysaccharide formation
• Urease enzyme + Inositol assimilation +
• Brown colonies produced on bird seed agar

92. Cryptococcus gattii – a closely related
relative of C. neoformans
• Isolated from forested area of the Pacific Northwest
(British Columbia, Washington, Oregon and California)
found in soil debris and tree species
• Infection of normal and immune suppressed hosts
• Mostly Pulmonary disease [Cryptococcoma] but can
develop meningitis like C. neoformans
• Culture and staining identical to C. neoformans except for
L Canavanine glycine bromthymol blue medium –
C

93. Positive India Ink
Urea medium demonstrating urease
enzyme activity of Cryptococcus
Observe
Budding cells
Variability in size
Positive
Mucoid colonies of
C. neoformans and
C. gattii

94. C. neoformans/C. gattii forms
brown colonies on Birdseed agar

95. Mucicarmine stain
Stains the capsular polysaccharide of capsule
Pneumocystis jiroveci could
be confused with C.
neoformans – Careful!
Central nuclear staining
C. neoformans/ C. gattii

96. CUTANEOUS AND
SUPERFICIAL
MYCOSES
Malassezia furfur
Dermatophytes

97. Malassezia furfur
• Pityriasis versicolor
• Most superficial of the dermatomycoses
• Found as normal flora on the skin,
• More common on oily skin or high use of skin oils
• Diseases:
• Skin: macules, papules, patches, plaques on
chest back and shoulders with either hypo or
hyper pigmentation – does not invade into deeper
tissues
• Fungemia in neonates caused by skin flora
tunneling in the IV lipid feeding lines

98. Malassezia furfur
• Lipophilic yeast – oil required for growth
• Media used for culture must contain oil or have oil
overlay
• Small budding yeast 2 – 4 µM with collarette
Spaghetti and meatballs

99. Size range for Yeast
• Candida glabrata/Histoplasma capsulatum
• 2 – 4 um
• Candida species
• 8 – 10 um plus pseudohyphae
• Cryptococcus neoformans/gattii
• 2 – 20 um
• Blastomyces dermatitidis
• 8-15 um

100. Dermatophytes – Ringworm
infections
• Hair, skin and nail infections
• 3 genera of fungi
• Microsporum species (many)
• Epidermophyton floccosum
• Trichophyton species (many)
• Disease described by area of the body infected:
tinea capitis (head), t. pedis (foot)
• Usually a clinical diagnosis not requiring culture
• Can do a KOH prep or Calcofluor white prep to
visualize fungal hyphae

101. Positive KOH prep
Showing thin septate
fungal hyphae
Calcofluor white stain
with fluorescence –
thin fungal hyphae

102. Microsporum canis
Main cause of ringworm from dog and cat
White colony/ yellow on backside of colony
Tuberculate macroconidia [spiny projections]
Few if any microconidia

103. Microsporum gypseum infection from
exposure to contaminated soil

104. Trichophyton
rubrum
White colony with
red diffusable
pigment
Pencil shaped
Macroconidia
Many micro-
Conidia
Infection from
fomites
Red diffusible pigment

105. Trichophyton
tonsurans
Ballooning
Microconidia
Primary cause of
epidemic scalp
ringworm in
children from
infection of Hair

106. Epidermophyton floccosum
Beaver tail large spores without microconidia
Khaki green colony

107. Opportunistic Fungal
Pathogens
Infections in the immune suppressed host
or special circumstances
Hyaline molds
Black molds

108. Opportunistic Fungi - hyaline
• Hyaline – no color to the hyphae
• Regular septations in the hyphae
• Branching – angle can be helpful in identification
• Usually grow in 3 – 5 days at 30˚C
• ??? of species thousands– taxonomy changing
daily

109. Aspergillus species
• Hyaline with septations
• Numerous round conidia
• In tissue - Branching at 45 degree angle
• Primarily pulmonary infection in immune suppressed
• Invade vessels, cause thrombosis & infarctions

110. Aspergillus Structure

111. Aspergillus species
Four species most common in human infections:
1. Aspergillus fumigatus
2. Aspergillus flavus
3. Aspergillus niger
4. Aspergillus terreus – unique and important – only
Aspergillus species resistant to Amphotericin B
Aspergillus Galactomannan Enzyme immunoassay –
detects circulating Aspergillus antigen in the blood
and/or bronchial lavage fluid
Problems with low sensitivity and specificity
False positive reaction in patients on therapy with
Piperacillin/Tazobactam, infected with Histoplasma
capsulatum

112. Aspergillus fumigatus
Blue/Green colony
Phialids with spores are
Directed upward

113. Aspergillus flavus
Green colony
Orange colored spores
that surround the vesecle

114. Aspergillus niger
Black colony
Black spores surround the vesicle

115. Aspergillus terreus Sandy colored colony
Aleurioconidia
Resistance to Amphotericin B

116. Aspergillus
•fruiting head dichotomous
•branching septate hyphae
• branching at 45* angle
Pseudallescheria boydii
can be confused for
Aspergillus, hyphae is a bit
thinner. Culture separates
the two fungi.

117. Fusarium species –
Common in nature/plants
disease related to immune
status of host
Infections reported:
Disseminated in bone marrow
transplants
Corneal infections in contact
lens wearers

118. Scopulariopsis species –
Soil and plants
Infections: Nail, skin, sinusitis, pulmonary and disseminated

119. Paecilomyces species
Isolated from soil and food
Opportunistic pathogen in the
immune suppressed

120. Penicillium species – most common mold
in the environment, bread mold, uncommon
cause of human disease

121. MUCORMYCOSIS/
ZYGOMYCOSIS
Hyaline
Broad non-septate hyphae

122. Mucormycosis/Zygomycosis
• Infections in diabetics, the elevated
glucose enriches fungal growth – classic
infection is rhinocerebral mucormycosis
• Sinus and pulmonary infection in the immune
suppressed host
• Broad, hyaline, aseptate hyphae produced
• Cultures grow in 24 hrs, coarse aerial hyphae
• Can be difficult to culture – tube like hyphae killed
during manipulation and plating
• Should not grind tissue
• Mince tissue and place on agar

123. Zygomycete – coarse, aerial hyphae after 24
hours growth on SABS agar at 30˚C

124. Rhizopus Absidia
Distant rhizoids
Mucor
No rhizoids
Rhizoids

125. 90˚ angle branching, aseptate, ribbon like
Invades vessels and can cause
infarcts and thrombi
Zygomycetes (Mucorales)