Mycology

• single, round-oval structure

• creamy colonies

  • “feet” extending from colony

• smells like bread

• see “budding” on stain

what characterizes yeast?

• filamentous fungi

• form mycelium and aerial hyphae

• fluffy, cottony colonies

what characterizes mold?

many hyphae intertwined

what is mycelium?

food-absorbing portion

what is vegetative hyphae?

grows above the surface—support reproductive structures

what is aerial hyphae?

an antifungal agent that inhibits contaminating fungi

what is cycloheximide?

with and without antibiotics and cycloheximide

what should a battery of fungal media have?

• Sabouraud’s Dextrose Agar

• BHI agar + blood, gentamycin, and chloramphenicol

• SABHI agar (SAB + BHI agar)

• inhibitory mold agar

what are some fungal medias?

chloramphenicol and gentamicin

what inhibits bacteria with the inhibitory mold agar?

dimorphic pathogenic fungi

what is the inhibitory mold agar a primary recovery of?

• specimen storage

  • process ASAP

  • refrigerate if the specimen is likely to be contaminated with normal flora

  • otherwise leave at room temp

• use biological safety cabinet

• fungi are BSL-3 organisms

• tape all fungal plates shut!!

how are fungal specimens processed?

• 1st am specimen best for 3 successive days

• bronchial washings

• bronchoalveolar lavage

• tracheal aspirations

• store at 4℃

• include at least one medium with cycloheximide

what characterizes a sputum specimen?

• 1st am specimen best

• clean catch

• storage: refrigerate

• process: centrifuge urine and use sediment

• media: need media with antibiotics

what characterizes a urine specimen?

include media with cycloheximide and antibacterial agents

what media is used for wounds/tissue specimens?

centrifuge: use sediment

how is CSF processed?

no antibacterial or antifungal agents

what type of media is used on CSF specimens?

dermatophyte culture

what type of culture is used on hair, skin, and nail scrapings?

place in sterile petri dish or paper envelope

how are hair, skin, and nail scrapings transported?

media that contains antibiotics and cycloheximide

what media is used for hair, skin, and nail scrapings?

• KOH

• Calcofluor White

• India Ink Prep

• Gomori Methenamine Silver stain

• Periodic Acid-Schiff Stain

• Wrights stain

what can be used on a direct exam of fungal specimens?

• dissolves proteinaceous material

• background is cleared and can see fungal elements

what is the function of KOH?

• emulsify specimen in 10% KOH

• coverslip

• read after 10-15 mins

what is the procedure of KOH?

fungal cell walls

what does Calcofluor bind to?

1. mix specimen with KOH + Calcofluor White

2. Remove excess fluid

3. examine with fluorescent microscope

4. blue-white or green fluorescence

what is the procedure of Calcofluor White?

Cryptococcus sp.

what does India Ink Prep identify?

halo or clearing around cell

what is looked for with the India Ink Prep?

• fungi stain dark brown to black

• background is usually green

what characterizes the Gomori Methenamine Silver Stain?

fungi stain bright red-purple against blue background

what happens with the Periodic Acid-Schiff Stain?

• 25-30℃

• incubate for 4 weeks

what is the incubation conditions of fungal cultures?

• tease mount

• scotch tape prep

• microslide culture

what are the microscopic identification methods from cultured mold colonies?

• two needles are used to remove a portion of the colony

• add LactoPhenol Cotton Blue to the slide with the organism

  • phenol kills organism

  • lactic acid preserves fungal structures

  • Cotton Blue stains the chitin in the cell wall

what is a tease mount?

• scotch tape pressed onto fungal colony

• lay the tape, sticky side down, on LPCB

what is scotch tape preparation?

• inoculate block of agar with organism

• coverslip and incubate in humidified petri dish

• remove agar block; add LPCB and examine

what is the slide culture method?

long strands of cells

• tubular filaments develop from spores

what are hyphae?

crosswalls

what is septate?

no crosswalls

what is nonseptate?

lightly pigmented; shades of blue or green

what characterizes hyaline?

hyphae and conidia have natural dark natural dark pigmentation

what characterizes dematiaceous or phaeoid?

asexual reproductive structure

what is conidia (spore)?

stalk on which the conidia develop

what is a conidiophore?

small, unicellular spores

what is microconidia?

large, usually multiseptate; club or spindle shaped

what is macroconidia?

spores formed directly from the hyphae by fragmentation through the points of septation

what are arthroconidia?

round, thick-walled resistant spores

what are chlamydospores?

• found in yeasts

• the budding portion of multiplying yeast

what is blastoconidia?

• filaments that are pinched in at the point of attachment

  • “sausage links”

• produced by some yeast

what is pseudohyphae?

include media that have cycloheximide and chloramphenicol added to inhibit bacterial organisms and saphrophytic fungi

A sputum specimen from a patient with a known Kleb. pneumoniae infection is received in the lab for fungus culture. The proper procedure for handling this specimen is to:

• reject the specimen and request a repeat culture when the bacterial organism is no longer present

• incubate culture tubes at room temperature in order to inhibit the bacterial organism

• include media that hav cycloheximide and chloramphenicol added to inhibit bacterial organisms and saphrophytic fungi

• perform a direct PAS stain; if no fungal organisms are seen, reject the specimen

Calcofluor White

A smear of skin tissue reveals fluorescent septate hyphae. The smear was prepared using:

• acridine orange

• calcofluor white

• auramine rhodamine

• periodic acid-schiff

Cycloheximide

The recovery of some Cryptococcus species may be compromised if the isolation media contains:

• cycloheximide

• gentamicin

• chloramphenicol

• penicillin

true

T/F: Mycetoma is a chronic infection involving the subcutaneous tissue, skin, and contiguous bone caused by bacteria and fungi.

• opportunistic

• systemic

• subcutaneous

• superficial

what are the types of mycosis?

• mold or yeast

• septate or non-septate

• hyaline or dematiaceous/phaeoid

what are the possible morphologies of fungi?

• Aspergillus sp.

• Penicillium sp.

• Mucormycetes

what are some opportunistic mycoses?

widely found in nature

where are Aspergillus sp. found?

• pulmonary disease

• inhalation of spores

• colonize old lung cavities (fungus ball)

• invasive

what characterizes Aspergillosis?

• narrow and septate

• dichotomous branching (45° angles)

how does Aspergillus hyphae appear?

• “closed fist” conidia structure

• has an afro

how does Aspergillus conidia appear?

• common contaminant

• rare pathogen

what characterizes Penicillium sp.?

• fungal eye infection

• trauma to cornea

• may require corneal transplant

what characterizes Mycotic Keratitis?

“open fist” conidia structure

how does the conidia of Penicillium appear?

Zygomycetes

what was Mucormycetes previously known as?

found worldwide on decaying vegetable material or in soil

where are Mucormycetes found?

• inhalation of spores

• vascular invasion

• rhinocerebral form

• infects GI tract

what characterizes Mucormycosis?

fast grower—fills containers

what type of growers are Mucormycetes?

• branching

• predominantly non-septate

• broad, irregular, ribbon-like hyphae

what is the hyphae of Mucormycetes?

formation of a sporangium with sporangiospores

what is asexual Mucormycetes reproduction?

formation of a sporangium from zygospores. Requires + / - hyphae strains

what is sexual Mucormycetes reproduction?

capable of producing single-cell and multi-cellular forms (pseudohyphae and hyphae)

what are yeast-like fungi?

• Candida

• Cryptococcus

• Torulopsis

• Trichosporon capitatum

what falls under the yeast-like fungi category?

throat, GI tract, skin

where is Candida albicans normal flora?

• Candidiasis

• Thrush

• Vaginitis

• Bronchopneumonia

what diseases are caused by Candida albicans?

1. microorganism + serum at 35℃ for <3 hours

2. prepare wet mount and examine microscopically

3. germ tube = filamentous extension from cell (formation of true hyphae)

what are the steps to a germ tube with Candida albicans?

• inoculate microorganism by making parallel cuts into agar

• place sterile coverslip on site

• incubate for 24-48 hours

• look for: Chlamydospores, blastoconidia, and pseudohyphae

what is the procedure of cornmeal agar?

C. albicans: Chlamydospores, blastoconidia, and pseudohyphae

what is the interpretation of the cornmeal agar?

no

is Candida auris normal flora?

direct contact with contaminated surfaces, equipment, or individuals

how is Candida auris transmitted?

multidrug-resistant organism

what type of organism is Candida auris?

sepsis, wounds, and ear infections

what can Candida auris cause?

• Sabouraud Salt Dulcitol Broth for isolation

• MALDI-TOF or molecular testing required for identification

what is used to identify Candida auris since traditional testing methods are difficult?

it does not produce pseudohyphae

what is different about Candida auris compared to other Candida sp.?

light blue with blue halo; blue from the back side

how does C. auris appear on CHROMagar Candida Plus?

green blue

how does C. albicans appear on CHROMagar Candida Plus?

metallic blue with pink halo

how does C. tropicalis appear on CHROMagar Candida Plus?

muave

how does C. glabrata appear on CHROMagar Candida Plus?

pink and fuzzy

how does C. krusei appear on CHROMagar Candida Plus?

white or pink

how does C. auris appear on original CHROMagar?

wild birds

• pigeon droppings

what is the habitat of Cryptococcus neoformans?

• pulmonary

• meningitis

what diseases are caused by Cryptococcus neoformans?

halo due to polysaccharide capsule

how does Cryptococcus neoformans appear with India Ink prep?

• CSF

• Serum

• Urine

what specimens are used in antigen detection of Cryptococcus neoformans?

• latex beads coated with antibody to Cryptococcus

• test of choice—much better than India Ink prep

• if test positive—perform titer

what characterizes the antigen detection of Cryptococcus neoformans?

media without cycloheximide

what type of media is used to culture Cryptococcus?

phenol oxidase production

• enzyme + dihydroxyphenylalanine→melanin

what does Birdseed agar test for?

brown colonies

how does Cryptococcus appear on birdseed agar?

white colonies

how does Candida appear on birdseed agar?

• growth at 37℃

• cycloheximide “S”

• urea +

• nitrate =

• pseudohyphae =

• true hyphae =

• arthroconidia =

what are the identification tests of C. neoformans?

in trees

• tropical/subtropical regions

what is the habitat of Cryptococus gattii?

respiratory route

how is Cryptococcus gattii transmitted?

healthy and immunocompromised hosts

who can be infected with Cryptococcus gattii?

masses of lesions on lung & brain

what is Cryptococcus gattii associated with?