mycology

1. MLS 302 C - MICROBIOLOGY 3
MYCOLOGY
VIROLOGY

2. PRELIMS OUTLINE
• General characteristics of fungi
• Morphology of fungi
• Classification of fungi
• Laboratory Diagnosis of Fungal
Diseases
• Classification of Fungal Diseases

3. REFERENCES
• Jawetz, Melnick & Adelberg’s Medical
Microbiology
• Clinical Mycology by Dismukes, et al.
• Bailey & Scott’s Diagnostic Microbiology
• Clinical Microbiology Made Ridiculously
Simple

4. M Y C O L O G Y

5. LECTURE 1: OBJECTIVES
• 1. Define terms used in mycology
• 2. Discuss the general
characteristics of fungi
• 3. Describe the morphology and
structure of fungi in general
• 4. State the medical importance of
the cellular structures of fungi

7. FUNGAL
CHARACTERISTIC
• Eukaryotic
• Facultative anaerobic/strictly aerobic
• Chemotrophic; nutrition by absorption
• Non-photosynthetic; achlorophyllos
• pH 5-6 (acidic)
• High sugar conc’n. favors growth

8.  Widely
distributed in
nature
 Saprophytic

9. Gastronomic
Delights

10. Penicillium notatum
Tolypocladium inflatum

11. Phytopathogen

12. Mycoses

13. TERMINOLOGY
• Mykos (Gk): Mushroom
• Mycology: Study of fungi
• Mycoses: diseases caused by fungi
• Dimorphism: ability to grow as yeast at
37°C and as mold at room
temp.
• Hyaline fungi: non-pigmented fungi
• Dematiaceous fungi: pigment-producing fungi

14. Morphologic Classification
of FUNGI
 YEAST
 MOLD
 DIMORPHIC

15. YEAST
Unicellular fungi
• MICROSCOPIC
 Oval to round
 Budding
 Bud=Blastospore
 Pseudohyphae

16. BUDDING YEAST

17. • MACROSCOPIC:
Pasty, opaque, cream-colored colonies

18. • MONOMORPHIC
YEAST:
 Candida
albicans
 Cryptococcus
neoformans
 Geotrichum
candidum

19. MOLD
 Multicellular
 Hypha [2-10 µm]
 MYCELIA
 Vegetative /
Thallus
 Reproductive /
Aerial

20. MOLDS: Mycelia

21.  MACROSCOPIC:
Cottony, wooly,
velvety, granular,
filamentous
 MONOMORPHIC
MOLD:
• Microsporum
• Epidermophyton
floccosum
• Trichophyton

22.  PIGMENTATION:
observed from the reverse

23. A. EXISTENCE OF SEPTA
 CLASSIFICATION OF HYPHAE

24. In Medically Important Fungi:
3 Types Of Hyphae
1. Coenocytic- sparsely
septated
2. Dematiaceous fungi -
dark and pigmented
septate hyphae
3. Hyaline molds: septate,
non-pigmented hyphae

25. B. HYPHAL SHAPES

27. DIMORPHIC FUNGI
Thermal dimorphism (a group of pathogenic fungi)
Mold form: 25ºC to 30ºC
Yeast form 35ºC to 37ºC

28. DIMORPHIC FUNGI
• Sporothrix schenckii
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis
• Paracoccidioides brasiliensis
• Penicillium marneffei
Subcutaneous
Systemic
Opportunistic

29. CAPSULE
Structure: Polysaccharide
Functions: Antiphagocytic factor
Cryptococcus neoformans (encapsulated yeast)

30. CELL WALL
• Antigenic
• Multilayered
a. polysaccharides
 poorly degraded by host
 activate CF; provokes inflammatory rxn
 Induce immune hypersensitivity
b. proteins and glycoproteins

32. CELL WALL
Functions:
 Provides shape, rigidity & strength
 protection from osmotic shock
 mediates attachment to host cells

33. Major Polysaccharides Of Fungal
Cell Wall
POLYMER MONOMER
Chitin N-acetyl glucosamine
Chitosan D-Glucosamine
Cellulose D-Glucose
-Glucan D-Glucose
-Glucan D-Glucose
Mannan D-Mannose
The type & amount of polysaccharide vary
from one fungal species to another.

34. CELLULAR MEMBRANE
Bilayered Structure:
1. Phospholipids
-phosphatidylcholine
-phosphatidylethanolamine
2. Sterols
- ergosterol
- zymosterol

35. CELLULAR MEMBRANE
Functions:
 Protects cytoplasm
 regulates intake & secretion of
solutes
 facilitates capsule & cell wall
synthesis

36. • CYTOPLASM
Nucleus, nuclear membrane, nucleolus,
ER, mitochondria, vacuoles

37. LECTURE 2: OBJECTIVES
• Classify fungi
– according to methods of reproduction
– type of spores they produce.

38. REPRODUCTION
A. SEXUAL REPRODUCTION
 opposite gametes mate to form a zygote
 Sexual spores
3 phases of sexual reproduction:
1. Plasmogamy – fusion of opposite but
compatible mating types
2. Karyogamy – fusion of 2 nuclei to form a
diploid nucleus
3. By meiosis, diploid nucleus gives rise to haploid
spores

39. SEXUAL REPRODUCTION

40. SEXUAL SPORES
ZYGOSPORE
• Enclosed in a thick wall
• Rhizopus, Mucor

41. ASCOSPORE
 Produced in ascus
(sac-like); 2-8
spores/ascus
 Nucleic fusion of
morphologically
dissimilar cells
 Histoplasma
capsulatum

42. BASIDIOSPORE
formed externally on a
base pedestal called
Basidium
 mushroom

43. ZOOSPORE
Fusion of cells from
2 diff. hyphae
OOSPORES
• thick-walled, long-
surviving spore
Phytophthora
capsici

44. B. ASEXUAL REPRODUCTION

45. SPORE GERMINATION
 Fruiting body : Principal structure
 asexual spores

46. SPORE DISPERSAL

47. CONIDIOSPORE
•Unicellular / multicellular
•Produced in a chain at the
end of conidiophore
•Penicillium
•Aspergillus
ASEXUAL SPORES

48. A. Arthrospore
•fragmented septate
hyphae
• single, slightly
thickened cells
•Coccidioides
•Geotrichum

49.  bud
Candida
Cryptococcus
B.
Blastospore
C. Chlamydospore
• Thick walled
• formed along the
periphery or tip of the
hyphae

50. SPORANGIOSPORE
•developed in a sac
(sporangium)
•Attached to a specialized
hyphae (sporangiophore)

51. C. PARASEXUAL REPRODUCTION
 Set of events that lead to genetic
exchange via mitotic recombination
 Initiated by formation of a heterokaryon
(thallus w/ 2 haploid nuclei of 2 diff.
genotypes)
 for genetic analysis of imperfect fungi

53. FUNGI-TAXONOMIC
CLASSIFICATION
• Depends primarily on the type of
sexual spore
Phylum -mycota
Class -mycetes
Order -ales
Family -ceae
Genus
Species

54. Fungi-Taxonomic classification
SEXUAL SPORE CLASS
Zygospore---------- Zygomycetes
Basidiospore--------Basidiomycetes
Ascospore---------- Ascomycetes
Zoospores---------- Chytridiomycetes
None/Unknown---- Deuteromycetes
(“Fungi Imperfecti”)

55. ZYGOMYCOTA
sporangium fungi / common
molds
molds & blights
(Rhizopus stolonifer)
 coenocytic
 rhizoids

57. ASCOMYCOTA
• sac fungi
• Reproduction:
–Conidiospores
–Ascospores
–Budding
• Ascocarp
• Ascus

59. BASIDIOMYCOTA
• club fungi
• Some are used as
food; others cause crop
damage
• Seldom reproduce
asexually

60. • Basidiocarp
• annulus
• basidia
• Volva

62. DEUTEROMYCOTA
 Asexual spores: conidia of various types
 Septate hyphae
 most are yeast & molds, some dimorphic

63. LECTURE 3:
OBJECTIVES
Discuss the laboratory
methods used in the
diagnosis of fungal diseases
as to:
1. proper collection, handling,
transport and disposal of
specimen
2. culture media to use
3. methods of identification

64. SPECIMEN COLLECTION
• Aseptic technique
• Collect spx from actual infxn site
• Adequate quantity
• Accurate label; Prompt delivery

65. CLINICAL SPECIMEN
Blood 5 ml to BHI broth
Bone
Marrow
Aspirate 0.5ml to BHI
CSF Lumbar tap
 Vol. is >2ml: centri, smear,
culture
Vol. is <2ml: use uncentri spx.

66. • Direct culture
• Biphasic culture bottle
• Membrane filtration
• Lysis centrifugation

67. • Sputum
• Bronchial
washings
• Tracheal
aspirate
 Collect sputum in a.m. for 3
days
No 24 hr. collections
sterile NSS for washings
Throat
 + few yeast & contaminants
 Use 2 sterile swabs
 Use tongue depressor if ?
candida

68. Urine  Midstream, clean-catch,
catheter
 Centri, smear, culture
(sediments)
Process in 2hrs or refrigerate
>100,000/ml is significant
Vaginal &
cervical
discharges
 +few to moderate Candida
colonies
2 swabs: for KOH & culture

69. CUTANEOUS
a. Hair  May contain contaminants
 Pluck hair by roots (forceps)
 Select hair that fluoresce, broken,
scaly
b. Nail  70% alc., scrape discolored areas,
collect inner infected nail
 Nail clippings: cut into small pcs.
 w/KOH
c. Skin  Few Candida & contaminants
 scrape infected area
 w/KOH

70. Skin Scrapings Nail clippings

72. Muco-
cutaneous
Scrape plaque w/ TD
(Candida)
Subcutaneous Lesions, abscesses,
tissues
Needle aspiration or
biopsy
Check for granules

73. LABORATORY DIAGNOSIS OF
FUNGAL INFECTIONS
• Direct microscopic examination
• Culture
• Serology
• others

74. MICROSCOPIC METHOD
WET MOUNT
A. 10-20% KOH
 destroy keratin layer
 OH
o clears debris
o makes fungi
prominent

75. B. Lactophenol Cotton
Blue (Aman’s medium)
 Lactic acid
 Phenol
 Cotton blue

76. C. India Ink
 capsule of C.
neoformans
 Capsules
(clear halo)
against dark
background

77. D. Calcofluor White +
KOH
 For dermatophytes
 Not suitable for
Pneumocystis carinii
 Binds with chitin in cell
wall
 under Wood’s light/UV
light
 apple-green/ bluish-
white fluorescence

78. II. STAIN PREPARATION
•
TISSUE
SECTION
•Grocott’s
•Griffith’s
•Gomori’s
• Methenamine
Ag*
•PAS**
* Stains cell wall
black
** Stains hyphae of
molds & some
yeast
PIGMENTED
TISSUE
•Fontana
Tribondeau
MALIGNAN
T
CELLS
•PAPS •better demo of
B. dermatitidis
than wet mounts

79. BONE
MARROW
•Giemsa
•Wright’s
•Detects intracellular
H. capsulatum in
bld. smears
•Orgs. appear as
small, oval yeast cells
(light-dark blue)
GENERA
L TYPE
•Kinyoun’s AFS
•Hucker’s GS
• fungi are GS + but
poorly stained
• Useful for Candida

80. PAS
Grocott/Gomori/Methenamine
Silver Stain
Wright’s H&E

81. Gram stain

82. CULTURE METHOD
REQUIREMENTS
Media must include:
•Amino acids or urea (N), Glucose (C), etc.
• Antimicrobial agents (cyclohexamide &
chloramphenicol)
 Aerobic environment;  humidity & moisture

83. TYPE ADVANTAGES DISADVANTAGES
AGAR
PLATES
• Better aeration
• Large surface area
for better isolation
• Ease of handling
• Easily dehydrates (use
40 ml agar)
• biological safety
cabinet
• Hazardous to handle
SCREW-
CAPPED
TUBE
•Easy storage
•Less space for
incubation
•More easily handled
•Less hazardous
•Lower dehydration
rates
•Poor colony isolation
• reduced surface area
•Promote anaerobiosis

84. CULTURE MEDIA
PRIMARY CULTURE
 SDA – Sabouraud Dextrose Agar ; general isolation
 BHI – useful for pathogenic fungi from sterile
spx.
 SDA/BHI w/ antibiotics

85. SELECTIVE CULTURE MEDIUM
 corn meal agar
 corn meal agar w/ Tween 80 –
stimulates chlamydospore formation (Candida)
 CZAPEC agar – routine media for Aspergillus &
Penicillium
 Cottonseed agar - conversion of B. dermatitidis
mold phase to yeast phase
 Niger seed/ Birdseed agar – C. neoformans ;
brown– black colonies
 Rice agar
 Urea agar

86. III. CULTURE METHOD: Primary culture
Medium
1. Sabouraud Dextrose Agar (SDA)

87. III. CULTURE METHOD: Primary culture Medium
2. Brain Heart infusion Agar
3. SABHI + antibiotics
– Recovery of saprobic and pathogenic fungi

89. III. CULTURE METHOD: Primary
culture Medium
4. SDA/BHI w/ antibiotics
• Recovery of pathogenic fungi exclusive of
dermatophytes

90. III. CULTURE METHOD: selective medium
1. Corn meal agar
2. Corn meal agar w/ Tween 80

91. Cornmeal Tween80

92. III. CULTURE METHOD: Selective medium
3. Czapek agar
– Differential ID of Aspergillus spp.

93. III. CULTURE METHOD: Selective medium
4. Urea agar
– detection of C. neoformans
– differentiate T. mentagrophytes vs T. rubrum
– detection of Trichosporon spp.

94. Culture method: selective
5. Niger seed/
Birdseed agar
– Identification of C.
neoformans
– Phenoloxidase

95. Culture method: selective
6. Cottonseed conversion agar
– conversion of B. dermatitidis from mold to yeast
form
7.Rice agar
– ID of M. audouini

96. IV. GERM TUBE TEST
• Candida albicans
• 0.5 mL rabbit serum +
a colony
• incubate for 2 ½ - 3
hours @ 37°C
• (+) Germ tube
formation

97. V. Hair baiting/perforation
• T. mentagrophytes
• Fill petri dish w/ soil then make
holes on soil
• Cut hair into small pcs. & place
in holes
• Incubate @ RT
• Examine regularly for growth.

98. VII. REDUCTION OF NITRATE TO NITRITE
• Cryptococcus neoformans

99. BIOCHEMICAL TEST
CHO Assimilation Test For Yeast
 (+) growth around disk
Reduction Of Nitrate To Nitrite
 C. neoformans
Rapid Urease Test
 For preliminary ID of C. neoformans

100. EXAMINATION OF FUNGAL GROWTH
MACROSCOPIC
• Rate of growth
• Topography – flat, heaped, folded, rugose,
wrinkled
• Texture – cottony, velvety, powdery, creamy or
pasty
• Pigmentation – surface & reverse side

101. Rate of Growth
• Rapid or slow grower
• Specify days or weeks

102. Topography
• Flat, heaped or folded
• Rugose: deep furrows that radiate from
the center
• Umbonate: elevated in the center
• Wrinkled or verrucose

103. Texture
• Cottony or woolly
• Velvety or silky
• Powdery or granular
• Moist, creamy, pasty

104. Pigmentation
• Surface pigmentation and pigmentation
on the reverse side
• Color description is very specific

105. EXAMINATION OF FUNGAL GROWTH
MICROSCOPIC (to observe spores & conidia)
• TEASE MOUNT – rapid method
- 1 gtt LPCB + sample
• SLIDE CULTURE
• CELLOPHANE TAPE MOUNT – scotch tape
mtd.

107. Tease Mount (double layer tape
prep)

108. SLIDE CULTURE
• Most accurate method to preserve & observe fungi
• Allows fungi to be preserved in orig. state
• Requires more skill & time than tease mount
Not for:
•Histoplasma,
•Coccidioides,
•Blastomyces,
•Sporothrix,
•Paracoccidioides

109. 028

110. SEROLOGICAL TESTS
• Used to supplement microscopic &
culture mtds.
• Methods:
– Agglutination
– Precipitation
– Complement Fixation (CF)
– ImmunoFluorescence (IF)
– Enzyme Linked Immunosorbent Assay
(ELISA)

111. SEROLOGICAL TESTS
Antigen Detection
 May hamper cross
reacting components
 Cryptococcal Ag test
–detects PS Ag from
CSF (latex agglut’n.)

112. Antibody Detection
 Relies on correct timing of spx collection
 Detects active infxns. for Histoplasmosis,
Blastomycosis, coccidioidomycosis,
sporothricosis
 May be hampered by cross reacting
antigens

114. OTHER TESTS

118. Classification of Fungal
Diseases
• Superficial
• Cutaneous
• Subcutaneous
• True systemic (endemic)
• Opportunistic

121. Superficial mycoses
Disease Etiological
Agent
Symptoms Identification of organism
Pityriasis
versicolor
Malassezia
furfur
hypopigment
ed macules
"spaghetti and meatballs"
appearance of organism in
skin scrapings
Tinea
nigra
Exophiala
werneckii
black
macules
black, 2-celled oval yeast
in skin scrapings
Black
piedra
Piedraia
hortai
black nodule
on hair shaft
black nodule on hair shaft
composed of spore sacs and
spores
White
piedra
Trichosporum
beigelii
creme-
colored
nodules on
white nodule on hair shaft
composed of mycelia that
fragment into arthrospores

122. Superficial Mycoses
• Pityriasis versicolor
– Chronic mild infxn of
stratum corneum
– Cosmetic problem
• Discrete, serpentine,
hypo- or hyperpigmented
spot on the skin (chest,
upper back arms or
abdomen)
• Enlarge and coalesce

123. M. furfur
hyperpigmented hypopigmented

124. Superficial mycoses
• P. versicolor
– Lipophilic yeasts
– KOH and calcouflour white
– Lesion flouresce under wood’s lamp
– Tx: selenium sulfide, topical or oral azole
– Implicated in seborrheic dermatitis or dandruff

125. Superficial mycoses
• Tinea nigra ( tinea
nigra palmaris)
– Asymptomatic infxn
caused by
dematiaceous fungi
Hortae (Exophiala
werneckii)
– Warm coastal regions
and among women
– Dark (brown to black)
palm discoloration

126. Superficial mycoses
• Tinea nigra
– Branched, septate
hyphae and budding
yeast cells w/ melanized
cell wall
– Tx: keratolytic solutions,
salicylic acid, azole
antifungal drugs

127. Superficial mycoses
• Piedra
– Black piedra: hard,
nodular infxn of the hair
– White piedra: larger,
softer and yellowish
nodules of the hair
– Tx: hair removal, topical
tx
– Endemic in tropical
underdeveloped
countries

128. Cutaneous mycoses
Dermatophytoses – (ringworm, caused by
dermatophytes) affect keratin-containing
tissues such as hair, nails, and skin.
– cellular IR may be evoked, causing pathologic
changes in the host that may be expressed in the
deeper layers of the skin
Dermatomycoses- cutaneous mycoses caused
by other fungi, most often Candida

129. Cutaneous mycoses
Etiological agents:
• Epidermophyton spp – affects only skin and
nails
• Trichophyton spp – can affect hair, skin, or
nails
• Microsporum spp. – usually affects only
hair or skin

130. Cutaneous mycoses
Disease Symptoms Identification
Tinea capitis Lesion in scalp
presence/absence and
shape of micro- and
macroconidia in
scrapings from lesion
Tinea corporis trunk, arms,
legs
Tinea manus hand
Tinea cruris "jock itch" groin
Tinea pedis"athlete's
foot"
foot
Tinea unguium Infection of nails
Ectothrix infection of hair
shaft surface mycelium and spores on
hair shaftEndothrix infection of hair
shaft interior

131. Clinical Manifestation: Dermatophytes

132. Clinical Manifestation: Dermatomycoses

133. Candida: KOH mount

134. Superficial/Cutaneous mycoses
Methods of diagnosis: Calcoflour white

135. KOH
Skin scrapings Nail scrapings

136. Macroconidia and Microconidia
• Macroconidia
– Large, multi-septate,
club, oval, spindle-
shaped
– Cell wall is smooth or
echinulate
• Microconidia
– Small, unicellular
– Round, elliptical.
pyriform, tear-shaped

137. • Microsporum
– Large multicellular
macroconidia;
microconidia rarely
produced

138. • Trichophyton
– Predominant forms
are microconidia;
macroconidia
uncommon
– Do not fluoresce on
Wood’s lamp

139. • Epidermophyton
– Macroconidia only;
microconidia not
produced

140. Endothrix Ectothrix

141. Superficial/ cutaneous mycoses:
Methods of transmission

142. Superficial mycoses
• Prevention and control

143. Subcutaneous mycoses
• fungal infections beneath the skin.
• Sporotrichosis – Sporothrix schenckii
• Chromomycosis – Fonsecaea, Phialophora,
Cladosporium

144. Systemic mycoses
• fungal infections deep within the body that
affect many tissues and organs.
• Coccidiodomycosis – Coccidioides immitis
• Histoplasmosis – Histoplasma capsulatum
• Blastomycosis – Blastomyces dermatitidis
• Meningitis - Cryptococcus neoformans

145. Systemic mycoses
• Ergot poisoning - Claviceps purpurea
• psilocybin "poisoning" - Psilocybe cubensis,
Psilocybe mexicana , Psilocybe tampanensis
and Psilocybe atlantis
• Aflatoxin poisoning - Aspergillus flavus
• Mycotoxin poisoning (phalloidin and amanitin)
- Amanita phalloides

146. Opportunistic mycoses
• caused by normal microbiota or fungi that are
not usually pathogenic.
• infect any tissues, however, they are usually
systemic

147. Opportunistic mycoses
• AIDS patients susceptible to Cryptococcus,
Pneumocystis, and Penicillium infections.
• Mucormycosis - Rhizopus and Mucor.
• Aspergillosis - Aspergillus.
• Candidiasis- C. albicans, can be dermal, oral
(thrush) or vaginal.

148. END OF PRELIMS