Prt. 2 Week 1 Lecture 2 Laboratory Diagnosis of Fungal Diseases Flashcards
• Suspected areas initially cleaned or wiped with 70% alcohol to remove surface contaminants
SKIN SCRAPINGS and SWABS
a. Hair
Pluck hair by roots (forceps/tweezers), place epilated hairs in sterile Petri plate for transportation
Select hair that..
fluoresce, broken, scaly
Nail scrape____ areas, collect inner infected nail/keratin debris
Nail clippings: cut into small pieces
discolored
Skin
Firmly scrape infected area (…), use blunt scalpel, tweezers, or bone curette
place in sterile Petri plate for transportation
advancing border of lesions
SKIN SCRAPINGS and SWABS
Direct microscopy: _______ and/or_____
KOH wet mount and/or Calcofluor stained mount (if necessary)
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
dermatophytes
Epidermophyton, Microsporum, and Trichophyton
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
Mucocutaneous/ Subcutaneous
Specimens
______plaque (Candida), lesions and wounds, place into sterile saline/broth
_______of deep cysts or abscesses
Check for_____
Scrape
Needle aspiration
granules
LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS
Methods
• Direct microscopic examination
• Culture
• Biochemical tests
• Serology
• others
• Ideal for observing skin, hair, or nails
• dissolves keratin layer
• clears debris
• enhances visibility of fungi
10-20% КОН
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
Observing for encapsulated yeast
i.e.
India Ink
Cryptococcus neoformans
Capsule
Stain binds with chitin and cellulose apple-green/ bluish-white fluorescence
Calcofluor white stain
Observed using fluorescence/UV microscope
10% KOH may be added for dermatophytes
Not suitable for_____
Calcofluor white stain
Pneumocystis carinii
• For observation of budding yeast, hyphae, conidia & hyphal filaments;
primarily for vaginal specimens
Saline wet mounts
• Stains fungi (except Actinomycetes) magenta against a light pink or green background
• hyphae of molds & some yeast
Periodic acid-Schiff (PAS) [permanent stain]
Periodic acid-Schiff (PAS) [permanent stain]
• Stains fungi (except_____) magenta against a light pink or green background
Actinomycetes
• Appear Gram-positive or blue/violet
• Used primarily to observe yeast and pseudohyphae in clinical specimens
Gram’s stain [permanent stain]
• Stain partially acid-fast Nocardia
• Fluorescent auramine-rhodamine acid fast stain may also be used
Acid-fast stain (modified Kinyoun) permanent stain]
TISSUE SECTION stains
• Griffith’s
• Grocott’s
• Gomori’s methenamine silver (Ag) - stains cell wall black
• stains cell wall black
Gomori’s methenamine silver (Ag) -
PIGMENTED TISSUE
• Fontana-Tribondeau
• Fontana-Masson
MALIGNANT CELLS
• Papaniculau stain (better demonstration of Blastomyces dermatitidis than wet method)
• 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
• 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
