2. Lab Safety & Diagnosis of Fungal Infections

What are some safety practices/equipment that are essential when working with fungi? (3)

class II biosafety cabinet

enclosed electric incinerator

screw-top tubes instead of petri dish

What equipment is useful in identifying infected hairs?

Wood lamp emitting UV light >365 nm

How are hair samples inoculated?

placed directly into sterile petri dish, inoculated onto fungal medium & incubated @22-30C

How are skin and nail samples collected? What is one way these samples can be prepared for analysis?

scraping skin and cutting nails

KOH wet mount

Why is KOH used when analyzing skin or nail samples?

KOH breaks down tissue/dissolves keratin → easier to view dermatophyte

Describe the KOH preparation (% solution, parts of sample to KOH, method)

10-20% solution

equal parts sample to KOH

coverslipped & heated gently then cooled 15 minutes

What is the mechanism of KOH with calcofluor white?

dye binds to cellulose/polysaccharides present in chitin → apple-green or blue-white in UV light

How are skin & nail samples inoculated?

directly on the agar

How are blood & bone marrow samples prepared for fungal analysis?

lysis centrifugation system (releases organisms from cells)

How are CSF samples prepared for fungal analysis?

concentrate via centrifugation before inoculation

How are abscess, wound, or tissue samples prepared for fungal analysis?

mince & grind tissue

How are CSF, abscess, wound, or tissue inoculated?

by plating directly on agar

What color is the background stain for India ink? What is it used to visualize?

black

visualize capsules of yeast or bacteria

What are the 5 tissue stains?

periodic acid-Schiff (PAS), GMS, H&E, Giemsa, Fontana-Masson

What are the 3 general types of mycology media?

primary plating media

sporulation inducing media

biochem media for ID

What are some characteristics of Sabouraud Dextrose Agar (SDA)? (4)

non-selective

nutritionally poor medium

acidic pH (5.6)

used because early colony morphology were done using SDA

What are the other 2 primary fungal culture media?

PDA & BHI

What are the specifications for incubating fungi? (4)

room temp (30C) or 37C for dimorphic

maintain 4-6 weeks

examine 2x week for growth

incubation characteristics should be recorded

What are the 4 things you are observing when making a macroscopic fungal ID?

colony growth time

color

texture

pigment on reverse side of colony

What are the 4 things you are observing when making a microscopic fungal ID?

septate vs aseptate

hyaline vs dematiaceous

fruiting structure

type, size, shape, arrangement of conidia

How do you perform a tease mount?

put a drop of LPCB on glass slide

remove portion of fungal culture w/ sterilized needles & add to LPCB drop

gently tease apart hyphae vs sterilized needles

cover w/ coverslip & examin @ 100-400x

How do you perform a cellophane tape preparation?

press cellophane tape gently on surface of colony

press one side of tape onto slide w/ LPCB

stretch tape & lower into stain, affixing to other side

What is the purpose of using a slide culture for fungal ID?

to maintain the morphology of fungi

How do you perform a slide culture preparation?

agar block placed on agar plate or sterile slide

block is inoculated with fungal culture

sterile coverslip placed on the block

plate is incubated

drop of LPCB added to slide

coverslip removed form agar block & placed onto LPCB to examine

What are 5 miscellaneous tests for molds?

hair perforation test (triangle observed in hair follicle)

urease test

thiamine requirement

trichophyton agars

growth on rice grains

What are 4 miscellaneous tests for yeasts?

germ tube

carbohydrate assimilation

chromogenic substrates

cornmeal agar

What are 7 miscellaneous tests for yeasts? (Pt.3)

potassium nitrate assimilation

temperature studies

urease

1-3B-Dglucan detection (cell wall component)

galactomannan (component of aspergillus cell wall)

T2MR (for diagnosis of invasive candidiasis)

cryptococcal antigen

How is the skin test reactivity to fungal antigens performed?

by using antigen extract

only valuable if patient has a history of a nonreactive skin test

Why have cross-reactivity & interlaboratory discrepancies been reported?

because some Ab’s have similar structure to fungal Ab’s

What are some antifungal agents used (5)?

amphotericin B. (many side effects)

fluconazole (FLU)

itraconazole (ITRA)

posaconazole (POSA)

voriconazole (VORI)

What institute is responsible for antifungal susceptibility testing?

CLSI