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Fungi characteristics

-Eukaryotic
- Mold, yeast, or dimorphic
-150 species cause disease
-Wide range of infections

Lab ID

-Macroscopic and Microscopic morphological characteristics
-Carb and extracellular enzyme production(physiological characteristics)
-Clinical picture and epidemiology

Use hood for this type of fungi

filamentous/mold

Shrink seals or fungi tape are used on plates with these fungi

fungi with aerial mycelium

perform this on all unknown molds before cell culture

wet prep

do not set up slide cultures on this type of fungi

dimorphic fungi

autoclave contaminated materials at

121oC for 1 hour at 15 psi

BSL 1

agents not known to cause disease in health adults

BSL 2

agents associated with human disease or hazard; percutaneous injury, ingestion, mucous membrane exposure

BSL 3

indigenous or exotic agents with potential for aerosol transmission; disease may have serious consequences

BSL 4

dangerous or exotic agents which pose high risk of life threatening disease, aerosol transmitted lab infections, or related agents with unknown risk of transmission

Many fungal infections are similar to which other infection and therefore is often ordered with fungal cultures

mycobacterial infection

rapid transport and processing is important because of contamination by...

bacteria and saprophytic fungi

deliver to lab within in ____ hours of collection

2

set up fungal culture within__hours of receipt into lab

2

sources for specimens include

respiratory, CSF, blood, cutaneous,urine, tissue, bone marrow, sterile body fluids, urogenital, and wounds

This percentage of healthy women have yeast normally

20%

Routine incubation temperature

30oC

2 sets of cultures for dimorphs are incubated at_____and _______ oC

25 and 37

routine fungal cultures must be kept __days before being called negative

30

yeast fungal cultures

keep 7 days and discard

Rapid grower

less than 5 days

Intermediate grower

6 to 10 days

Slow grower

greater than 11 days

Moniliaceous

clear, colorless, pastel color

Dematiaceous

darker staining on the reverse (also maybe on the obverse)

yeast-like

looks like Candida albicans

glabrous

smooth

velvety

suede fabric, short uniform length

cottony

long aerial mycelium/hyphae

granular

sugary; reproductive structures are heavily sporulating

septate hyphae

have septations

aseptate hyphae

rare septa; large, wide, ribbon-like

hyaline

moniliaceous; clear/pale reverse color and hyphal elements pastel color

dematiaceous

dark, olive, black, brown reverse colony color

special formations seen mostly in dermatophytes

1. Raquet hyphae
2. nodular organs
3. pectinate bodies (comb)
4. favic chandeliers (antlers)
5. spiral hyphae
6. chlamydoconidia
7. arthroconidia

Storage units, swollen cells found in pseudohyphae, refractile, appear empty, may be intercalary between blastoconidia of pseudohyphae or terminal (on end of pseudohyphae)

Chlamydospores

Asexual reproductive cells produced directly form true hyphae, appear to have contents in structure

Chlamydoconidia

Cyclohexamide

anti fungal agent

Chloramphenicol

antibiotic

BHI (Brain Heart Infusion agar)

nutritious, used to get fastidious dimorphs growing ; can add blood or antibiotics

Sabouraud Brain Heart Infusion agar (SABHI)

able to grow fastidious fungi

SABHI with blood

able to grow fastidious fungi, more nutrients

Sabouraud Dextrose agar (SDA)

supports most fungal growth; 4% glucose or 2% glucose in the modified version

BHI biphasic blood culture bottles

typically inoculate a blood culture bottle if you have blood

You want to give enough_____to grow, but not too much that the fungi don't _______.

nutrients, sporulate

Inhibitory Mold Agar (IMA)

selective, primary isolation medial; doesn't grow dermatophytes, has chloramphenicol but not cyclohexamide

Dermatophyte Test Medium (DTM)

screen only, not definitive, pH indicator (phenol red); stimulates conidia formation and pigment formation

Potato Flake Agar

makes fungi sporulate

Mycosel or Mycobiotic agar

selective for saprophytes; contains cyclohexaminde and chloamphenicol, primary dermatophytes

Yeast extract phosphate agar

inhibits saprophytes but grows slow growing fungi

Mycosel

SDA with chloramphenicol and cycloheximide

Inhibitory mold agar

yeasts will grow on this but bacteria are inhibited

A good specimen for using BHI with blood is___

sputum

Ascospore agars

1. V8 Juice agar
2. Malt Extract agar

Corn Meal Tween 80 agar

used to ID Candida albicans; stimulates clamydospore production

Cotton Seed conversion agar

used for Blastomyces dermatidis; converts dimorphs from mold to yeast form

Czapek's agar

Aspergillus recovery and identification

Niger/Bird Seed agar

contains caffeic acid and used for ID Cryptococcus neoformans-turns brown

This enzyme causes C. neoformans' positive reaction on NSA

phenoloxidase

Nitrate reduction agar

helps ID nitrate reducing organismsm, esp. yeasts

Potato Dextrose Agar (PDA)

stimulates pigmentation and conidia formation

Rice Medium

Microsporum audouinii doesn't grow on this

Trichophyton agars 1-7

Different chemicals in all 7 agars; ID species; hard to read

Urea agar

salmon=neg
magenta pink=positive

Yeast fermentation broth

anaerobic, sugars fermented

Yeast Nitrogen base agar

contains oxygen, simulation?

CHROMagar

selective and differential for Candida species

Which organism is green on CHROMagar

Candida albicans

Direct exam benefits

allows a preliminary report to physician, may enable fungal therapy, may lead to special media inoculation, may prevent overtreatment

Saline wet mount used for to see...

budding yeasts
hyphae/pseudohyphae
conidia
fungus-like bacteria
granules
spherules in Coccidioides immitus

Lactophenol Cotton Blue (LPCB)

Phenol-kills organisms
Lactic acid- preserves fungal structures
Cotton blue-stains chitin in fungal structures to make them visible

Can lactophenol cotton blue be made permanent?

yes

KOH prep

-used for hair, skin, nails, sputum, vaginal, and bronchial washings when a lot of cellular material is present
-dissolves keratin

What is the purpose of the KOH prep dissolving keratin?

make fungi more visible

Is the KOH prep permanent?

no. but you can add glycerol to save for 2-3 days

What color do fungi and yeasts stain in the Gram stain?

crystal violet/blue (Gram positive)

Budding yeasts indicate ____

infection

Gram Stain

-Yeasts and pseudohyphae are the most common elements observed

In the Gram Stain, Cryptococcus neoformans may be overlooded in which 2 specimens?

CSF or respiratory specimens

Modified Kinyoun Acid Fast stain

-ID aerobic, branching GPRs or fungus like bacteria, especially Norcardia species
-uses weaker acid to decolorize: 1%H2SO4

Norcardia appears what color under Modified Kinyoun acid fast stain?

red. everything else counterstains blue

Kinyoun Acid Fast Stain

used for staining ascospores-look magenta/red
3% HCl Decolorizer

India Ink prep

Use blue/black ink, india ink, negrosin
used for observation of capsules, esp. Cryptococcus neoformans and other encapsulated yeasts appear positive

Calcofluor white stain

dye absorbed by chitin in the fungal cell wall
may be used in conjuction with 10% KOH

Observe this color fluorescence for Calcofluor white stain

bluish white or apple green (depending on wavelength)

Calcofluor white stain is not good for these specimens

vaginal specimens or Coccidioides spherules

Gomori methenamine silver (GMS)

histology; stains fungi and actinomyces; stain black with green background

Periodic acid Schiff (PAS)

fungal elements stain magenta against a pink or green background

H&E

background pink
fungal elements stain purple

Wright Stain

Hematology; look for Histoplasma capsolatum in blood or bone marrow; stain purple

Fontana-Masson Stain

rare; dematiaceous if positive (melanin-like pigment stains positive)

What stain is better than India ink?

Mayers mucicarmine-stains capsle of Cryptococcus red, the background is yellow

Tease mount advantages

can be performed on a mature colony from a primary isolation plate; can be turned into a permanent mount

Tease mount disadvantage

rough teasing may disrupt conidia and make structural morphology difficult to see

Cellophane tape mount advantage

preserves the integrity of conidia and sturctures more than a tease mount

Cellophane tape mount disadvantage

tape acts as a coverslip, so you can't seal it for permanent mounting

Slide culture method (and advantages)

best method for observation of fungal structures; can be preserved for permanent mounting; two mounts can be preserved by permanent mounting

Slide culture advantage

fungal growth onto the slide and coverslip maintains the morphological position of structures to make ID easier

Slide culture disadvantages

Technical to set up; must wait for culture to grow after initial time of fungus isolation; Zygomycetes grow past the edge of the coverslip before formation of reproductive structures

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