Mycology Flashcards
Mycology Laboratory Methods
Direct examination
- Calcofluor white
- fluorochrome
- binds to cellulose and chitin in fungal cell wall
- highlights fungi, prototheca, and cysts of acanthamoeba
- India ink
- Fontana Mason stains capsule of cryptococcus
- GMS or PAS in tissue
Fungal culture
- Brain heart infusion (BHI) agar
- Sabouraud dextrose agar
- Inhibitory mold agar
- above agars incubated at 25-30 degrees for 4-6 weeks
- Special media:
- Cottonseed agar converts mold phase of blastomyces to yeast form
- cornmeal or potato dextrose agar (latter shows pigment form Trichophyton rubrum)
- cornmeal or rice agar with Tween 80
- Sabouraud dextrose agar, Dixon medium, or Leeming-Notman medium overlaid with sterile olive oil
- Trichophyton agars
- Bird seed (niger seed) agar
Sabouraud dextrose agar (SDA)
- principle
- purpose
- Principle
- Acid pH and high dextrose concentration inhibits bacterial growth but permits growth of fungi; some also contain abx
- Purpose
- many fungi
Inhibitory mold agar (IMA)
- principle
- purpose
- Principle
- chloramphenicol and sometimes gentamicin or ciprofloxacin to suppress bacteria
- Purpose
- selective isolation of fungi from specimens that may contain commensal bacterial flora
Brain heart infusion agar (BHI)
- principle
- purpose
- Principle
- brain/meat infusion, peptone and dextrose
- Chloramphenicol and gentamicin can be added
- Principle
- nonselective formulation is general purpose medium used for bacteria, yeasts, and molds
- when containing abx, it prevents commensal bacterial from growing
Cyclohexamide containing media
- principle
- purpose
- Principle
- inhibits growth of many saprophytic fungi, while permitting growth of most pathogenic fungi
- chloramphenicol and gentamicin can be added
- Purpose
- Selective isolation of slow growing pathogenic fungi that may be overgrown by rapidly growing saprophytic fungi
- cycloheximide also inhibits growth of C neoformans, Candida, Aspergillus, and other zygomycetes
- targets dermatophytes or thermally dimorphic fungi
Olive oil overlay on SDA, Dixon medium, or leeming-notman medium
use
isolate malassezia
Trichophyton agars
differentiate between species of trichophyton
Bird seed agar
demonstrate pheno oxidase activity of cryptococcus neoformans
Mold morphology
- Hyaline septate molds
- surface of colonies may be white or colored
- reverse side of plate is usually light
- hyphae with frequent septations
- Dematiaceous molds: septate molds that make melanin
- surface and reverse side of plate are both dark
- Zygomycetes: aseptate or pauciseptate
- lid lifters
- may be pigmented, but do not make melanin (reverse side is light)
- Dimorphic fungi: septate hyphae at 25-30 degrees; yeast when reincubated at 37 degrees
Yeast gross morphology and classification
- creamy or mucoid (cryptococcus) colonies; molds make fuzzy colonies
- candida albicans form “feet” starlike projections
- further classified by MALDI or by special yeast agar
Yeasts with blastoconidia only
Cryptococcus
Candida glabrata
Rhodotorula
Malassezia
Yeasts with blastoconidia and pseudohyphae
Candida (except glabrata)
Saccharomyces cerevisiae
Yeast with arthroconidia
Trichosporon
Geotrichum
Hyaline molds
Hyphae and other structures are nonmelanized
- aspergillus
- penicillium
- fusarium
- dermatophytes
Dematiaceous molds
Alternaria
P boydii/S boydii
Scedosporium prolificans
Curvularia
Mold with pauciseptate hyphae
Zygomycetes (rhizopus, mucor, cunninghamella, rhizomucor)
Thermally dimorphic fungi
Histoplasma
Blastomyces
Coccidioides
paracoccidioides
sporothrix
penicillium marnefii
Histoplasma capsulatum
- diagnosis
- culture
- geographic distribution
- transmission
- appearance in tissue
Diagnosis and culture
- 2-4 um ovoid yeast with narrow based budding
- in histiocytes
- slow growing cottony white mold
- septated hyaline hyphae with intermittent microconidia and large spiny macroconidia
- undergo yeast conversion at 37 degrees
Ohio and Missippi river valleys, Latin America, and Spain
Primary infection is pulmonary, can be disseminated (reticuloendothelial); sclerosing mediastinitus
Transmitted via droppings from chickens or bats
Histoplasma duboisii
- geography
- disease
- culture
- tissue
- Africa
- local or disseminated (skin, soft tissue, bone)
- Colony and microscopic morphology indistinguishable from var capsulatum
- often intraceullar within giant cells
- round to oval, thick walled yeast, narrow based budding (unlike B dermatitidis which is a similar size)
Blastomyces dermatitidis
- diagnosis
- culture
- disease
- similar morphology to ___
- geographic distribution
- transmission
- uniform, large yeasts (8-15 um) with broad based budding
- thick wall and double contoured yeast
- Slow growing mold with cottony white surface that darkens to tan with age at 25-30 degrees
- septate hyaline hyphae with short, unbranched conidiophores producing single, pyriform to round, smooth conidia (lollipops)
- Morphology similar to nondimorphic mold Chrysosporium
- Conversion to yeast at 37 degrees
- Mississipppi and Ohio River valleys; also in southeastern US and around Great Lakes
- soil, inhaled
- pulmonary primary infection
- disseminated infection affects skin, mucous membranes, and bone
Coccidioides immitis/posadasii
- morphology
- resemble ___
- yeast conversion?
- geographic distribution
- transmission
- disease
- large spherules with thick walls that contain hundreds of endospores
- 25-30 degrees grows as moist gray colonies that become white and cottony as they mature
- thin, hyaline septate hyphae and arthroconidia; mature arthroconidia are barrel shaped, and alternate with empty cells
- Immature arthroconidia resemble Malbranchea
- difficult yeast conversion
- San Joaquin Valley California (C posadasii in southwest)
- soil inhaled
- Can be easily inhaled by lab personel (be careful!)
- primary infection is pulmonary
- disseminated infection affects skin and bone and joints
paracoccidioides brasiliensis
- morphology
- gross
- yeast conversion
- geographic distribution
- disease
- round, large yeast with circumferential budding
- 25-30 degrees: slow growing mold with a white to tan surface
- hyaline septate hyphae with terminal and intercalary chlamydospores and infrequent pear shaped microconidia arranged along hyphae
- converts to yeast at 37 degrees
- Central and South American rain forest
- primary infection is pulmonary
- Can disseminate: skin, mucosa, reticuloendothelial
- Behaves like blastomyces
Sporothrix schenkii
- morphology
- geographic distribution
- transmission
- disease
- 4-6 um, cigar shaped yeast with narrow based budding
- 25-30 degrees: grows rapidly into moist, white to pale orange colonies that turn brown with age
- delicate hyaline septate hyphae producing conidiophores topped by clusters of microconidia (rosettes or daisies)
- converts to yeast at 37 degrees (cigars with narrow based buds)
- worldwide distribution
- acquired by penetrating injury from contaminated plant (rose garden)
- Alcoholics known to become infected through inhalation
- lymphocutaneous infection with nodular, ulcerative lesions following lymphatics
Penicillium marneffei
- appearance of clinical sample
- culture
- disease; geographic distribution
- 3-5 um yeast divide by fission in clinical sample
- Culture
- 25-30 degrees: rapidly growing tan colonies initially powdery or velvety on the surface, and become colored with maturity (blue/green centrally)
- red pigment diffuses into agar around colonies
- hyaline septate hyphae with conidiophores and metulae producing brushlike clusters of phialides
- converts to yeast at 37 degrees (3-5 um), oval
- penicilliosis: endemic in southeast Asia
Aspergillus species
- clinical specimens appearance
- culture appearance
- clinical
- hyaline, septate hyphae with 45 degree branching
- fruiting heads in air filled tissue pockets
- Culture:
- rapidly growing; identified by colony morphology and microscopic appearance of swollen vesicle at ends of conidiophores
Culture appearance of aspergillus fumigatus
- blue green with white apron and light reverse
- conidiophores terminate in a swollen vesicle having a single row of phialides that cover only the top 2/3 of the vesicle
- each phialide gives rise to chain of small conidia
Culture appearance of aspergillus flavus
- yellow green to olive with light reverse; lateral striations
- circumferential phialides
- some strains uniseriate (single row of phialides) or biseriate
Aspergillus niger morphology
- Dark brown to black colonies with light reverse
- vesicles with 2 rows of phialides (biseriate) covering the entire vesicle
- phialides produce chains of rough, round, dark conidia
Aspergillus terreus morphology
- colonies are cinnamon brown on the surface with a yellow or orange reverse
- phialides cover top 2/3 of vesicle , but unlike fumigatus are biseriate and have longer chains of conidia
Diagnosis of aspergillus
- Culture
- ELISA for serum marker galactomannan and/or 1-3-beta-D-glucan
Aspergillosis
- transmission
- clinical disease
- clinical features of A flavus, A niger, and A terreus
- Ubiquitous in soil and on decaying vegetables
-
Respiratory tract affected
- Immunocompetent host will have cavitary lung disease: aspergilloma (fungus ball); most commonly caused by A fumigatus
- Atopic host: allergic bronchopulmonary aspergillosis (ABPA) or allergic sinonasal aspergillosis (allergic fungal sinusitis)
- Immunosuppressed host: invasive bronchopulmonary aspergillosis (IBPA) or invasive fungal sinusitis, with angioinvasive growth (Aspergillus species and zygomycetes have capacity to invade vessel walls)
- A flavus produces aflatoxins in food, carcinogens for HCC
- A niger pulmonary infection associated with oxalosis (calcium oxalate tissue deposition); most common aspergillus associated with otitis externa
- A terreus resistant to amphotericin B
Fusarium infections
similar to aspergillus + fungal keratitis and opportunistic infection in burn wounds
Pseudallescheria boydii/Scedosporium boydii disease
- transmission
- diseases
- treatment
- most common cause of eumycotic mycetoma after penetrating trauma
- fungal keratitis
- pneumonia after near drowning accidents
- P boydii/S boydii resistant to amphotericin B
Conidia arranged in clusters
Fusarium
- canoe shaped
- multicellular macroconidia with 3-6 cells each
Acremonium
- long, threadlike, unbranched phialides bearing clusters of single celled microconidia
Gliocladium
- branching conidiophores bearing flask shaped phialides, resembling Penicillium; microconidia cluster in a ball (golfball at end of finger tip)
Conidia arising in chains
Penicillium
- brushlike arrangement of flask shaped phialides that give rise to unbranching chains of microconidia
Paecilomyces
- Branching conidiophores with elongated, flask shaped phialides arranged in pairs or brushlike groups
- long chains of oval or spindle shaped microconidia bend away from the phialides
- colonies are yellowish brown or pinkish mauve, white, or yellow-green, but are never bright green or blue-green
Scopulariopsis
- single or branched conidiophores that give rise to annellides (similar to phialides)
Conidia arranged singly
Chrysosporium
- single, cutoff microconidia directly on hyphae or on the tips of simple conidiophores
- Looks like blastomyces
Sepedonium
- singly borne, hyaline conidia at the ends of branched or unbranched conidiophores
- conidia are large, thick walled and echinulate (spiny), resembling macroconidia of mold form of H capsulatum
Beuveria
- single, small, round or oval microconidia emerging from each inflection point in a zigzagging (geniculate) flask shaped conidiophore
Dermatophytes are grown on what medium?
cycloheximide
Diagnosis of dermatophytes
- KOH prep or calcofluor white prep of skin scrapings
- Species ID by colony morphology (trichophyton rubrum produce red pigment on reverse)
- Species ID by microscopic examination of conidia
-
Trichophyton rubrum:
- Tear shaped microconidia along hyphae: “birds on a telephone wire”
-
Trichophyton mentagrophytes:
- microconidia are arranged in clusters
- spiral hyphae
- cigar shaped macroconidia
- can penetrate hair shafts, unlike T rubrum
- Trichophyton tonsurans: microconidia have marked variability in size and shape; scalp ringworm
-
Microsporum canis:
- Macroconidia are spindle shaped and rough (echinulate) and pointed
- Macroconidia contain > 6 cells separated by transverse septae
-
Microsporum gypseum:
- Macroconidia are oval with rounded ends unlike M canis and have transverse septae
- Macroconidia contain less than 6 cells
- Microconidia also present
-
Epidermophyton floccosum:
- Macroconidia are smooth, club shaped found singly or in clusters
- Macroconidia contains 2-6 cells, separated by transverse septae
- No microconidia
Microsporum canis
Trichophyton mentagrophytes