MYCO Flashcards
Study of fungi
MYCOLOGY
fungi are
a. eukaryote
b. prokaryote
a. Eukaryote (have true nucleus)
characteristic of fungi that became the reason why nourishment must come from environment
Achlorophyllous (lack chlorophyll)
fungi are
a. anaerobes
b. facultative anaerobes
c. obligate aerobes
c. obligate aerobes
fungi that said to inhabit water, soil and decaying organic debri
Saprophytic
fungi are said belong to plant kingdom without roots and stems
Thallophytes
composition of fungi’s cell wall
Chitin
*resembles keratin as to function (provides protection) but not as to composition
composition of fungi’s cell membrane
Ergosterol
required for fungi for growth
moisture (↑ humidity)
fungi are
a. fast growers
b. slow growers
b. slow growers
T/F
Fungi may exhibit monomorphism or dimorphism
T
Existing only as yeast or mold
Monomorphic
Existing either as yeast or mold
Dimorphic (temp. dependent)
2 BASIC MORPHOLOGICAL FORMS OF FUNGI
yeast
mold
Unicellular morph form of fungi
YEAST
Morph form of fungi that mostly reproduce asexually by budding
YEAST
colonies of yeast
moist creamy, opaque, pasty
(resembles bacterial colonies)
what is required for yeast ID
physiologic/biochem tests since most yeast spp. has similar microscopic and colonial morphology
morph form of fungi that grows at 37C
yeast
morph form of fungi that grows at room temp
mold
Multicellular, filamentous morph form of fungi
MOLD
colonies of mold
fluffy, cottony, woolly or powdery
fundamental microscopic unit of fungi
Hyphae
Function of hyphae
absorption of nutrients from environment
mass of countless hyphae
Mycelium
PARTS of mycelium
Vegetative mycelium (thallus)
Aerial mycelium (reproductive)
part of mycelium that penetrates the surface medium, absorbs nutrients
Vegetative mycelium (thallus)
part of mycelium that extends above the surface; contains fruiting bodies that produces spores
Aerial mycelium (reproductive)
structures for reproduction
spores
Types of Hyphae
Aseptate (coenocytic)
Septate
Spiral (coiled hyphae)
Nodular bodies
Racquet
Pectinate body
Favic chandelier/antler
no cross walls
Aseptate (coenocytic)
divided into cells by crosswalls
Septate
ex. of aseptated
Zygomycetes (group Zygomycota)
ex. of septated
All except Zygomycetes
Spirally coiled type of hyphae
Spiral (coiled hyphae)
type of hyphae with club-shaped areas
Racquet
Enlargement in the mycelium
that consists of closely twisted
hyphae
Nodular bodies
type of hyphae that is short, unilateral projections from hyphae; broken-comb like
Pectinate body
type of hyphae that is curved, freely branching; resembles antlers of the dee
Favic chandelier/antler
ex. of fungi with spiral or coiled hyphae
Trichophyton mentagrophytes
ex. of fungi with nodular bodies
Microsporum canis
ex. of fungi with racquet hyphae
Epidermophyton floccosum
ex. of fungi with pectinate body
Microsporum audouinii
ex. of fungi with Favic chandelier/antler
T. schoenleinii
T. violaceum
fungi that produces both sexual and asexual spores
Synanomorphs/polymorph (perfect fungi)
fungi that produces only 1 type of spores
Imperfect fungi
fungi that produces only SEXUAL spores
Teleomorph
fungi that produces only ASEXUAL spores
Anamorph
process of SEXUAL spores formation
meiosis; result of nuclear fusion
process of ASEXUAL spores formation
mitosis; without nuclear fusion
enumerate sexual spores
Ascospores
Basidiospores
Zygospores
Oospores
Spores formed within ASCUS (SAC-LIKE structure); Produced in a fixed number of 8
Ascospores
Spores formed within BASIDIUM (CLUB-SHAPED structure)
Basidiospores
Formed from the union of 2 undifferentiated/identical hyphal cells
Zygospores
Formed from the union of 2 differentiated/non-identical hyphal cells
Oospores
enumerate ASEXUAL spores
Conidia
Sporangiospores
asexual spores produced singly or in groups attached by specialized vegetative hyphal strands called conidiophores
Conidia
unicellular, pyriform, elliptoid shaped conidia
Microconidia
larger, multicellular, spindle-shaped conidia
Macroconidia
enumerate conidia
Arthroconidia
Blastoconidia
Chlamydoconidia
conidia formed through hyphae fragmentation
Arthroconidia (Arthrospore)
conidia formed through budding
Blastoconidia
(Blastospore)
conidia formed from hyphae enlargement
Chlamydoconidia
(Chlamydospore)
ex. of Arthroconidia (Arthrospore)
Trichosporon
Geotrichum
Coccidiodes
ex. of Blastoconidia (Blastospore)
Candida
ex. of Chlamydoconidia (Chlamydospore)
Candida
parts of chlamydoconidia
Intercalary - within
Sessile - sides/lateral
Terminal - end
formed within a sporangium
Sporangiospores
ex of Sporangiospores
Zygomycetes
4 groups of fungi accdg to phylum
Zygomycota
Ascomycota
Basidiomycota
Deuteromycota
aka Conjugation fungi
Zygomycota
aka Sac fungi
Ascomycota
aka Club fungi
Basidiomycota
aka Fungi imperfecti
Deuteromycota
genera under Zygomycota
Mucor
Rhizopus
Absidia
genera under Ascomycota
Aspergillus (opportunistic)
Saccharomyces
Histoplasma capsulatum (causes systemic mycoses)
genera under Basidiomycota
C. neoformans (opportunistic)
genera under Deuteromycota
Dermatophytes
Fusarium
T/F
Spx collection for fungal ID thru swabbing is ACCEPTABLE
F
inadequate
All spx for fungi ID are refrigerated for a short period in case of delay except:
skin specimen
blood
CSF
spx collected using a fine needle; inoculated at bedside for fungal detection
Corneal scrapings
spx collected by aspiration; use of cotton swabs may give false + microscopic results
Pus
Most common specimen for fungal detection and ID
Respiratory tract secretions (sputum, bronchial washing & lavage)
Respiratory tract secretions are collected to recover these types of fungal agents
fungal agents causing systemic mycoses like H. capsulatum
spx for suspected fungal meningitis
CSF
CSF is for the detection of fungal meningitis caused by
C. neoformans (opportunistic, in immunocompromised)
how is CSF for fungal ID processed?
either thru filtration or sedimentation
pore diameter used to filter CSF for fungal ID
0.45 um
sedimentation process of CSF for fungal ID is done thru
centri
Sediments – for smear prep and culture
Supernatant – for serology
spx for fungal ID to r/o disseminated infections
Blood
spx that relies on the use of BACTEC
blood
specific type of fungi recovered using lysis centrifugation system of BLOOD
dimorphic fungi
a process performed in BLOOD spx for fungi ID that is necessary because many fungi are intracellular
lysis centrifugation system
allowable transportation duration of vaginal & urine spx
within 24 hrs
vaginal secretions are specifically screened for
yeast
temp and days of vaginal secretion incubation
30°C for 7 days
spx for the recovery of dermatophytes
Hair, Skin, Nails
Hair, Skin, Nails are spx used to recover and detect..
dermatophytes (causes cutaenous mycoses)
How is hair collected for dermatophytes recovery
Infected hairs may be plucked using forceps. Those hairs that fluoresce under Wood’s lamp may be selectively plucked. Hairs may be collected in sterilized paper envelopes
How is skin surface lesion collected for dermatophytes recovery
Skin surface must be disinfected with spirit before collection. The advancing edge of the lesion is scraped with the help of a blunt forceps and collected in sterilized paper envelopes
How is nail collected for dermatophytes recovery
Discolored or hyperkeratotic areas of nail may be scraped or diseased nail clipping may be collected in sterilized paper envelopes
how are Tissue, Bone Marrow, Sterile Body Fluids processed for fungi detection? what is the reason for doing that?
minced or use high speed blender
REASON: obtain cytoplasmic tissue content
where are tissue samples inoculated for fungi ID
inoculated on agar, NOT ON BROTH
Simplest method for direct examination of specimen for fungal ID
MICROSCOPIC EXAMINATION
T/F
Direct examination of specimens could be stained or unstained.
T
used to observe clinical specimens for the presence of fungal elements or to identifify the fungus following culture
Microscopy
enumerate TEMPORARY mounts
10-20% KOH
Lactophenol Cotton Blue (LPCB)
India Ink/ Nigrosin
Calcofluor white stain
enumerate PERMANENT mounts
Gram stain (Hucker modif.)
Periodic Acid Schiff (PAS)
Gomori Methenamine silver (GMS)
Acid Fast (Kinyoun’s)
Giemsa or Wright’s
Acridine orange
Fontana Masson
H&E
Mayer’s Mucicarmine
Gridley’s stain
Dissolves non-fungal materials - make fungal structures visible
NOT for sterile spx
10-20% KOH
a clearing agent; heat can increase clearing rate
10-20% KOH
KOH conc for skin and hair
10%
KOH conc for nails
20%
temporary mount for hair, skin, and nails
10-20% KOH
both a preservative and stain
Lactophenol Cotton Blue (LPCB)
Most widely used staining method for fungi
Lactophenol Cotton Blue (LPCB)
Components of LPCB and their function
Lactic acid – preservative
Phenol – killing agent
Cotton blue – stain
used to demonstrate capsule of C. neoformans
India Ink/ Nigrosin
a negative stain → result: colorless
*only the bg is stained, not the organism
*in histopath: it is an intravital stain
India Ink/ Nigrosin
used to demonstrate chitin
Calcofluor white stain
Best stain for direct microscopy
Calcofluor white stain
a fluorchrome dye for chitin demonst.
Calcofluor white stain
result of fungi in gram stain
gram +
what is hucker modification
(V[+AO] IAS)
- Crystal Violet + ammonium oxalate
- Iodine (mordant)
- Alcohol/acetone (decolorizer)
- Safranin (counterstain)
Stains fungi in tissues/histologic sec.
Periodic Acid Schiff (PAS)
Gomori Methenamine silver (GMS)
demonstrate details of fungal elements; a histologic stain
Periodic Acid Schiff (PAS)
In PAS
color of tissue with fungi and bg
Tissue w/ fungi: pink-red, bright magenta
Bg: green
used to demonstrate melanin
Gomori Methenamine silver (GMS)
GMS is used to detect these fungi
Candida
Aspergillus
BEST permanent mount
Essential for tissue pathology
Gomori Methenamine silver (GMS)
why is GMS essential for tissue pathology
Candida and Aspergillus may be missed in H&E
in GMS,
what is the color of outline, internal, and bg
Outline: Brown to black
Internal: Pink black
Bg: Light green
stain used to detect Blastomyces dermatitidis (agent of systemic mycoses) and Nocardia (branching bacteria)
Acid Fast
(Kinyoun’s)
stain to demonstrate yeast of H. capsulatum
Giemsa or Wright’s
wright’s stain is specifically used for:
giemsa stain is specifically used for:
Used in hematology:
Wright’s – blood cells
Giemsa – blood parasite
stain to demonstrate Malassezia furfur (agent of tinea/pityriasis versicolor – “an an”)
Acridine orange
Using acridine orange,
what is the color of M. furfur and epithelial cells
Fungi (M. furfur): Green fluorescence
Epithelial cells: Orange
Stains that detects hyphal pigmentation
Fontana Masson
H&E
Stains that differentiates hyaline from dematiaceous
Fontana Masson
H&E
nonpigmented or lightly pigmented structure using fontana masson and H&E
Hyaline
darkly pigmented structure using fontana masson and H&E
Dematiaceous
stain used to demonstrate C. neoformans
Mayer’s Mucicarmine
stain that stains hyphae and yeasts dark blue-pink
Gridley’s stain
what is the resulting colors if Gridley’s stain is used?
Hyphae, yeasts: Dark blue-pink
Tissues: Deep blue
Bg: Yellow
incubation temp of plates for fungi culture
ambient air at 25-30°C (RT)
culture for fungi ID is held for?
30 days
*should not be discarded for 4-6 weeks (slow growers)
fungi are handled using what class of BSC
Class II BSC
common antibacterial agents added in at least 1 culture medium for fungi to prevent contaminant rapid overgrowth
Chloramphenicol (inhibit bacterial growth)
Cycloheximide (for saprophytic fungi)
process that is demonstrated in vitro for dimorphic fungi ID
Mold → yeast phase
T/F
Fungi are identified on the basis of colony morphology (including pigmentation) and microscopic observation by tease-mount preparation or slide culture technique
T
general purpose media for fungi
Sabouraud’s dextrose agar (SDA)
media used for initial isolation of fungi
SDA
pH of SDA
5-6
SCREENING media for dermatophytes
Dermatophyte test medium (DTM)
indicator used by DTM
phenol red
media used for dermatophytes recovery
Mycosel or Mycobiotic agar
*DTM is for screening dermatophytes
composition of Mycosel or Mycobiotic agar
SDA + chloramphenicol + cycloheximide
Stimulate chlamydospore formation of C. albicans
Cornmeal agar
media for isolation of Aspergillus
Czapek agar
media for C. neoformans isolation
Niger seed agar/Bird seed agar or Staib’s medium
resulting color of C. neoformans in Niger seed agar/Bird seed agar or Staib’s medium? it is due to?
BROWN pigment due to phenol oxidase
media used to demo T. rubrum (+) red pigment production from T. mentagrophyte (–)
Potato dextrose agar
Media used to differentiate M. audouinii (- growth) from M. canis (+ growth)
Rice medium
a biochem test media → UREASE TEST
Urea agar
agar used to differentiate T. mentagrophytes (+) from T. rubrum (-)
ID Cryptococcus, Trichosporon, and Rhodotorula spp.
Urea agar
General media for yeast spp
Brain Heart Infusion agar
media for Nocardia
Casein Medium
media for M. furfur
Malt extract agar
media for the recovery of saprobic and pathogenic fungi
Potato flake agar
NOT a fungal media
Hay infusion agar
done to supplement microscopy and culture of fungi
Special tests
enumerate special tests for fungi
Hair perforation or Baiting test
Rapid Urease test
L-DOPA ferric citrate test
Germ tube test
Exoantigen test
Temperature studies
Carbohydrate Assimilation test
what organisms are differentiated in hair perforation/baiting test? what are the results?
T. mentagrophytes (+)
T. rubrum (-)
positive result of hair perforation/baiting test
V-shaped/conical hair perforation
*microscopic observation of hair strands
what is differentiated in Rapid Urease test? what are the results?
T. mentagrophytes (+)
T. rubrum (-)
Candida krusei (+)
All Candida are urease (-)
positive result of rapid urease test
Pink to purple color
positive and negative control used in rapid urease test
Positive control = C. neoformans
Negative control = C. albicans
test used for RAPID ID of C. neoformans (produce phenol oxidase)
L-DOPA ferric citrate test
L-DOPA is used for ID of what fungi
C. neoformans
positive result of L-DOPA ferric citrate test
Black color
Presumptive test for C. albicans
*Germ tube test
Germ tube test is for what fungi
C. albicans
C. dubliniensis
How is germ tube test performed?
✓ Transfer loopful of colonies into the tube
✓ Add 0.5 mL serum
✓ Incubate at 37C for 3 hours
✓ Transfer a drop into the slide → perform MICROSCOPY
positive result of germ tube test
Germ tubes appearance (resembles sperm cells; finger/hyphae-like extension of young yeast cells)
A microdiffusion test for serologic confirmation of systemic fungi (+) result presence of precipitin bands
Exoantigen test
positive result of exoantigen test
precipitin bands
what fungi are detected in exoantigen test
confirmation of systemic fungi
- Cocciodes immitis
- Histoplasma capsulatum
- Blastomyces dermatitidis
Exoantigen test:
antigens in presence of Coccidiodes immitis
CF, TP, HL
Exoantigen test:
antigens in presence of Histoplasma capsulatum
H or M band (both)
Exoantigen test:
antigens in presence of Blastomyces dermatitidis
A band
fungi observed using temperature studies? what is the positive result?
C. albicans (+)
C. dubliniensis (–)
C. neoformans (–)
Growth at 42°C
For ID of yeast isolate which is free from carbohydrates
Carbohydrate Assimilation test
Manual process of Carbohydrate Assimilation test
Nitrogen base agar (plated)
Place yeast suspension on agar surface
Allow to dry
Place comm. prepared disk w/ impregnated carbohydrate
Incubate at 30C for 24 hr
positive result of Carbohydrate Assimilation test
Growth around the disk
*Indicative of the yeast capability to utilize a particular carbohydrate as a sole source of carbon
general techniques for fungi ID
microscopic
culture
serology
antigen detection
skin tests
molecular techniques
Detection of this is helpful in diagnosis of sub-cutaneous and systemic mycoses, prognosis, and response to anti-fungal drugs
anti-fungal antibody (serology)
Different serologic techniques for anti-fungal Ab detection
agglutination
immunodiffusion
counterimmunoelectrophoresis
complement fixation test
immunofluorescence
RIA
ELISA
Particularly useful in the diagnosis of cryptococcal meningitis from CSF spx.
ANTIGEN DETECTION
fungi detected using SEROLOGICAL TESTS
- Blastomyces (agent of systemic mycoses)
- Histoplasma (agent of systemic mycoses)
- Cryptococcus (opportunistic)
- Aspergillus (opportunistic)
ANTIGEN DETECTION of fungi is performed by using these tests
Latex Agglutination or Immunodiffusion tests
Helpful in the detection of Aspergillus and Candida antigens in systemic infections
ANTIGEN DETECTION
used to demonstrate delayed hypersensitivity rxn to fungal antigens
skin tests
T/F
A positive skin test does not necessarily indicate an active infection; it only indicates sensitization of the individual
T
a fungi detection technique that has an epidemiologic value than diagnosis
skin tests
* positive skin test does not necessarily indicate an active infection; it only indicates
sensitization of the individual
Skin tests may be performed in these cases
Histoplasmosis
Candidiasis
Sporotrichosis
Coccidioidomycosis
Blastomycosis
Paracoccidiodomycosis
Dermatophytosis
A newer molecular technique for fungi ID
DNA hybridization
useful in dx of mycoses in a shorter period and detect those fungi that are difficult or dangerous to cultivate in vitro
PCR
T/F
molecular techniques may not be available for all fungi
T
Rapid Urease Producers (Bacteria)
Proteus
Providencia
Morganella
negative urease test result
yellow/orange