Prt. 2 Week 1 Lecture 2 Laboratory Diagnosis of Fungal Diseases Flashcards by Kyra Catedral

• Suspected areas initially cleaned or wiped with 70% alcohol to remove surface contaminants

SKIN SCRAPINGS and SWABS

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a. Hair

Pluck hair by roots (forceps/tweezers), place epilated hairs in sterile Petri plate for transportation

Select hair that..

fluoresce, broken, scaly

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Nail scrape____ areas, collect inner infected nail/keratin debris

Nail clippings: cut into small pieces

discolored

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Skin

Firmly scrape infected area (…), use blunt scalpel, tweezers, or bone curette
place in sterile Petri plate for transportation

advancing border of lesions

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SKIN SCRAPINGS and SWABS

Direct microscopy: _______ and/or_____

KOH wet mount and/or Calcofluor stained mount (if necessary)

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SKIN SCRAPINGS and SWABS

Scrapings vs Swabs

culture onto SDA with
cycloheximide/actidione, chloramphenicol & gentamicin, or a DERMASEL, 26C, 4 weeks

culture onto SDA-CG, 26C, 4 weeks

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dermatophytes

Epidermophyton, Microsporum, and Trichophyton

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Tissues & biopsy specimens

Aseptically collected and kept moist with __________ for transport;

a portion in_____ for histopathology (H&E, GMS, PAS)

Should include both normal tissue and center & edge of the lesion; tease apart in a sterile petri dish:

mince/homogenize tissue material:

sterile saline/BHI broth

formalin

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Mucocutaneous/ Subcutaneous
Specimens

______plaque (Candida), lesions and wounds, place into sterile saline/broth

_______of deep cysts or abscesses

Check for_____

Scrape

Needle aspiration

granules

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LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS

Methods

• Direct microscopic examination
• Culture
• Biochemical tests
• Serology
• others

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• Ideal for observing skin, hair, or nails
• dissolves keratin layer
• clears debris
• enhances visibility of fungi

10-20% КОН

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For direct smears or stains of fungal isolates

• Tease mounts for permanent smears and scotch tape preps
______-kill any live organisms
______preserves fungal structures
______blue stains chitin blue

Lactophenol Cotton Blue (LPCB)
[Aman’s medium]

Phenol

Lactic acid

Cotton

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Observing for encapsulated yeast

i.e.

India Ink

Cryptococcus neoformans
Capsule

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Stain binds with chitin and cellulose apple-green/ bluish-white fluorescence

Calcofluor white stain

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Observed using fluorescence/UV microscope
10% KOH may be added for dermatophytes

Not suitable for_____

Calcofluor white stain

Pneumocystis carinii

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• For observation of budding yeast, hyphae, conidia & hyphal filaments;

primarily for vaginal specimens

Saline wet mounts

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• Stains fungi (except Actinomycetes) magenta against a light pink or green background

• hyphae of molds & some yeast

Periodic acid-Schiff (PAS) [permanent stain]

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Periodic acid-Schiff (PAS) [permanent stain]

• Stains fungi (except_____) magenta against a light pink or green background

Actinomycetes

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• Appear Gram-positive or blue/violet

• Used primarily to observe yeast and pseudohyphae in clinical specimens

Gram’s stain [permanent stain]

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• Stain partially acid-fast Nocardia

• Fluorescent auramine-rhodamine acid fast stain may also be used

Acid-fast stain (modified Kinyoun) permanent stain]

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TISSUE SECTION stains

• Griffith’s
• Grocott’s
• Gomori’s methenamine silver (Ag) - stains cell wall black

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• stains cell wall black

Gomori’s methenamine silver (Ag) -

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PIGMENTED TISSUE

• Fontana-Tribondeau
• Fontana-Masson

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MALIGNANT CELLS

• Papaniculau stain (better demonstration of Blastomyces dermatitidis than wet method)

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• Papaniculau stain (better demonstration of______ than wet method)

Blastomyces dermatitidis

BONE MARROW • Detects intracellular Histoplasma capsulatum in blood smears • as small, oval yeast cells (light to dark blue)

Giemsa Wright's

GENERAL TYPE • Useful for Candida • Yeast and hyphae

Hucker's Gram Stain Hematoxylin & eosin

CULTURE METHODS REQUIREMENTS • Media must include: •_____ or ____,_____ etc. • With or without______ to inhibit growth of contaminating molds and bacteria. • _______environment •______ humidity & moisture • Examine____ weekly • 26-30°C for molds; 35C for yeasts (4 weeks)

Amino acids or urea (N), Glucose (C), etc. antimicrobial agents (ex. cycloheximide, gentamicin, chloramphenicol, ciprofloxacin Aerobic 40-50% 3x

Culture • Better aeration • Large surface area for better isolation • Ease of handling

AGAR PLATES

Culture • Easily dehydrates (use 40 ml agar) • Biological safety cabinet • Hazardous to handle

AGAR PLATES

Culture • Easy storage • Less space for incubation • More easily handled • Less hazardous • Lower dehydration rates

SCREW-CAPPED TUBE

•Poor colony isolation •Reduced surface area •Promote anaerobiosis

SCREW CAPPED TUBE

Primary culture medium - For general isolation; pH is___

Sabouraud Dextrose Agar (SDA) 5.6

Primary culture medium • Reserved for ***skin, hair, nail specimens,*** ex. SABHI + antibiotics

SDA with cycloheximide and chloramphenicol

Primary culture medium • - inhibits yeast phase of dimorphic fungi What is the antibiotic?

Chloramphenicol

Primary culture medium • Very nutritious, supports growth of bacteria & fungi, incl. ***Histoplasma and Nocardia***

BHI agar (BHIA)

Primary culture medium a.______ - recommended for converting dimorphic fungi from the mold to the tissue (yeast) phase b._______ - very nutritious but inhibits growth of Nocardia • Supports growth of dermatophytes

BHIA with blood BHIA with blood, cycloheximide & chloramphenicol

Primary culture media

SDA SDA-CC BHI/BHIA

CULTURE METHOD: Selective medium • Demonstration of ***blastoconidia, pseudohyphae, arthroconidia, & chlamydospores*** in the identification of Candida species and other yeasts

Corn meal agar CMA Corn meal Tween 80 (CMT 80)

Selective medium • Differential identification of Aspergillus spp.

Czapek agar

Selective medium - demonstrates urease production; pinkish purple within 48 hours inoculation

Urea agar slant

Urea agar slant • Positive: • Negative:

Trichosporon Rhodotorula Cryptococcus Geotrichum Saccharomyces most Candida

used for the differentiation of Trichophyton species.

BCP (bromcresol purple milk) and SDA (solids glucose agar)

• Caffeic acid + Phenoloxidase = dark brown to black pigmented colonies • For identification of C. neoformans

Niger seed agar or Birdseed agar (Caffeic Acid Agar)

For identification of ***C. neoformans***

Niger seed agar or Birdseed agar (Caffeic Acid Agar)

• primarily recover pathogenic fungi exclusive of dermatophytes

Inhibitory mold agar

• Enhance sporulation and pigmentation; subculture medium

Potato dextrose agar (PDA)

• Identification of Microsporum audouinii

Rice agar

• conversion of B. dermatitidis from mold to yeast form

Cottonseed conversion agar

• Speciation of Trichophyton species

Trichophyton test agars

• Screening medium for dermatophytes

Dermatophyte test medium (DTM); Dermasel agar

• Recover dermatophytes

Mycosel agar

MACROSCOPIC •_____ - rapid or slow; specify days or weeks •_____ - flat, heaped, folded, rugose (deep furrows that radiate from the center), umbonate (elevated in the center), wrinkled or verrucose •______ - cottony, velvety, silky, powdery, granular, moist, creamy or pasty •_______ - surface & reverse side; specify color

Rate of growth Topography Texture Pigmentation

• Media: rabbit, fetal calf, human serum (0.5 mL) [then add yeast colonies] • Demonstrate germ tube production of C albicans • Incubate for 2 ½ to 3 hours @ 37C • POSITIVE: Appendage half the width, 3x-4x length of yeast, no point of constriction at origin

GERM TUBE TEST

GERM TUBE TEST • Media:… • Demonstrate germ tube production of____ • Incubate for_____ at____ • POSITIVE: Appendage half the width, 3x-4x length of yeast, no point of constriction at origin

rabbit, fetal calf, human serum (0.5 mL) [then add yeast colonies] C albicans 2 ½ to 3 hours @ 37C

• Isolate keratinophilic fungi from soil • Fill Petri dish with soil then make holes on soil; moisten medium with sterile distilled water; place sterile cut-hair strands (small pieces) in holes; cover entire plate with paper or leave in a dark place; incubate at room temperature (4 weeks) • Observe for growth. Pick one hair with visible growth. Subculture on SDA/Mycosel agar/PDA. Record macroscopic and microscopic findings. Identify.

Hair baiting technique

Hair baiting technique • Isolate_____ fungi from soil • Fill Petri dish with soil then make holes on soil; moisten medium with sterile distilled water; place sterile cut-hair strands (small pieces) in holes; cover entire plate with paper or leave in a dark place; incubate at room temperature (4 weeks) • Observe for growth. Pick one hair with visible growth. Subculture on_________. Record macroscopic and microscopic findings. Identify.

keratinophilic SDA/Mycosel agar/PDA

• Identify Trichophyton mentagrophytes and its variants

Hair Perforation Test for Dermatophytes

• Identify Trichophyton mentagrophytes and its variants

Hair Perforation Test for Dermatophytes

• Microsporum canis (positive after 14 days) • Place autoclaved, cut (1 cm) blonde pre-pubital hair in vial with 5ml sterile distilled water; inoculate fragments of the test fungal colonies; incubate at room temperature (4 weeks); remove hairs at intervals; examine microscopically in LPCB or aniline dye or marked localized areas of pitting & marked erosion (conical perforations). • T. mentagrophytes - positive; T. rubrum - negative

Hair Perforation Test for Dermatophytes

EXAMINATION OF FUNGAL GROWTH MICROSCOPIC (to observe spores/conidia/hyphae) •_______ - rapid method; 1 drop LPCB + test fungal sample •________ - scotch tape or double-sided tape method •________

LPCB-TEASE MOUNT CELLOPHANE TAPE (cellotape flag) prep SLIDE CULTURE

Prt. 2 Week 1 Lecture 2 Laboratory Diagnosis of Fungal Diseases Flashcards

(62 cards)