Week 1 Lecture 2 Laboratory Diagnosis of Fungal Diseases Flashcards
SPECIMEN COLLECTION (review)
Important points:
a) Sterile technique
b) Adequate quantity
c) Sample from the area most likely
affected
d) Accurate label
e) Prompt delivery
Purpose:
• These specimens are collected when fungal septicemia (bloodstream infection due to fungi) is suspected.
• Special media and ______ are required for the best fungal recovery.
Blood & Bone Marrow Aspirates
Du Pont isolator tubes
Blood & Bone Marrow Aspirates
Procedure:
1. Collection of Blood
• Collect____ of blood from the patient.
• Centrifuge the sample and collect the____
• Prepare multiple smears for different staining techniques: (3)
• The remaining buffy coat is inoculated into culture media.
- Bone Marrow Aspirates
• The initial sample is used for___ staining.
• ____of the remaining bone marrow and blood is inoculated into culture systems.
5-10 ml
buffy coat
• Giemsa stain
• Gram stain
• Periodic Acid-Schiff (PAS) stain
Giemsa
3-5 ml
Purpose:
• Used for the concentration and culture of fungi from blood and cerebrospinal fluid (CSF).
• Superior technique for detecting fungi in these specimens.
Membrane Filter Technique
Membrane Filter Technique
Procedure:
1. The specimen is treated with (2).
• This lyses blood cells, releasing any fungal organisms.
2. The lysed sample is filtered under vacuum using a 0.45 µm membrane filter.
3. The membrane containing fungal elements is placed directly onto culture media for incubation.
Triton-X and sodium carbonate solutions
Purpose:
• Method of choice for processing blood cultures suspected of containing fungi.
• Breaks down host cells to release and concentrate fungal organisms.
Lysis-Centrifugation Isolator System (Wampole Isolator System)
Procedure:
1. The system contains a lysing agent that:
• Lyses white blood cells (lymphocytes) and red blood cells (erythrocytes).
• Inactivates plasma complement and certain antibiotics, which might interfere with fungal growth.
2. A centrifugation step is performed to concentrate fungi.
3. The concentrated sample is inoculated onto culture media.
4. Requires 10 ml of blood for optimal recovery.
Lysis-Centrifugation Isolator System (Wampole Isolator System)
Culture Systems for Blood & Bone Marrow Fungi Isolation
Direct Culture Method
• 0.5-1.0 ml of buffy coat (from centrifuged blood) is inoculated onto: (2)
• Brain Heart Infusion (BHI) slant
• BHI broth
Culture Systems for Blood & Bone Marrow Fungi Isolation
• Uses a biphasic bottle containing:
• BHIA slant (Brain Heart Infusion Agar)
• 60-100 ml of BHI broth
• The blood-to-broth ratio is 1:10 to 1:20.
• A minimum of 5 ml of blood is required.
• The bottle should be:
• Vented and tilted daily to allow oxygen exchange.
• Checked daily for fungal growth.
• Perform Gram staining to detect fungal elements.
Biphasic Filter Technique
BACTEC System
• BACTEC Fungal Medium is used for fungal culture.
• BACTEC MYCO/F Lytic Bottle contains antimicrobials to suppress bacterial contamination while allowing fungi to grow.
Primary Isolation Media for Blood & Bone Marrow Fungi (2)
• _________
• Two plates inoculated at 26°C and 35°C.
• ________
• Incubated at 35°C.
• Cultures should be maintained for______ to allow slow-growing fungi to develop.
Sabouraud Dextrose Agar (SDA) with Chloramphenicol & Gentamicin
Brain Heart Infusion Agar (BHIA) with 5% Sheep Blood
at least 4 weeks
Purpose:
• ____ is collected when fungal meningitis is suspected, commonly caused by Cryptococcus neoformans.
• Collected via_____
Cerebrospinal Fluid (CSF) Processing
lumbar tap (lumbar puncture).
Cerebrospinal Fluid (CSF) Processing
Procedure:
1. Collection and Transport
• Collect ____aseptically via lumbar puncture.
• Transport immediately; do not refrigerate.
• If storage is needed,_____
3-5 ml
keep at room temperature or incubate at 30°C
CSF
Processing
• Centrifuge the sample.
• Use___ for cryptococcal antigen testing.
• Use sediment for\_\_\_: • Place one drop on a slide and perform an \_\_\_\_preparation to detect Cryptococcus. •Culture ***1-2 ml of uncentrifuged or resuspended sediment*** onto: • \_\_\_\_\_\_\_\_ • Incubate duplicate cultures at 26°C and 35°C. • Maintain for at least 4 weeks. • \_\_\_\_\_\_\_\_ • Incubate at 35°C, maintain for at least 4 weeks.
supernatant
direct microscopy; India ink
Sabouraud’s dextrose agar (SDA) with chloramphenicol and gentamicin
Brain Heart Infusion Agar (BHIA) with 5% sheep blood
Respiratory Tract Specimens Processing
Types of Specimens:
• Sputum (Sputa)
• Bronchial washings
• Tracheal aspirates
• Throat swabs
Respiratory Tract Specimens Processing
Collection
• Best collected in the____.
• Transport in a sterile container, process immediately; store at___ for short delays.
• No____ collections (increases contamination risk).
• Use ____for washings.
• Use two sterile swabs with a sterile tongue depressor for throat collections.
• Sputa may require emulsification:
• Shake with ____sterile glass beads and____ sterile distilled water.
• Blood, pus, or necrotic material should be plated directly onto culture media.
• Few yeasts and contaminants are expected.
morning
4°C
24-hour
sterile normal saline solution (NSS)
12-20; 3-5 ml
Respiratory Tract Specimens Processing
Collection
• Best collected in the____.
• Transport in a sterile container, process immediately; store at___ for short delays.
• No____ collections (increases contamination risk).
• Use ____for washings.
• Use two sterile swabs with a sterile tongue depressor for throat collections.
• Sputa may require emulsification:
• Shake with ____sterile glass beads and____ sterile distilled water.
• Blood, pus, or necrotic material should be plated directly onto culture media.
• Few yeasts and contaminants are expected.
morning
4°C
24-hour
sterile normal saline solution (NSS)
12-20; 3-5 ml
Respiratory tract specimen processing
Laboratory Processing
• Prepare wet mount preparations using_____ and Gram-stained smears.
•____ may be used if the KOH mount is unsatisfactory.
• Culture inoculation:
• ___________
• Duplicate cultures at 26°C and 35°C.
•\_\_\_\_\_\_\_\_
• Incubate at 35°C.
• Maintain cultures for at least 4 weeks.
10% KOH
PAS stain
SDA with chloramphenicol and gentamicin
BHIA with 5% sheep blood
Urine Specimens & Vaginal/Cervical Discharges Processing
Procedure:
1. Urine Collection
• Collect midstream, clean-catch, or catheterized urine in a sterile container.
• Transport immediately to the lab.
• Centrifuge at 2000 rpm for 10-15 minutes.
• Use the sediment for staining and culture.
• Process within____ or _____
- Vaginal & Cervical Discharges
• Collect using two sterile swabs.
• Place into_____.
2 hours or refrigerate
transport media
Urine Specimens & Vaginal/Cervical Discharges Processing
Procedure:
- Laboratory Processing
• Perform______ mount for microscopy.
• Inoculate 0.05-0.1 ml onto_____.
• Duplicate cultures at 26°C and 35°C, maintain for 4 weeks.
potassium hydroxide (KOH) mount
SDA-GC (Sabouraud’s Dextrose Agar with Gentamicin & Chloramphenicol)
- Skin Scrapings
• Clean affected area with ____to remove contaminants.
• Scrape firmly using a_____
• Collect sample from the advancing_____
70% alcohol
blunt scalpel, tweezers, or bone curette.
border of lesions.
Hair Samples
• Pluck hairs by the____ using forceps or tweezers.
• Place in a____ for transport.
• Select hairs that______
root
sterile Petri dish
fluoresce, are broken, or have scaly surfaces.
Nail Clippings
• Scrape_____ areas and collect inner infected nail/keratin debris.
• Cut nail clippings into small pieces.
discolored
Skin, Hair, and Nail Specimens Processing
Laboratory Processing:
• Perform direct microscopy using_______ or ______(if necessary).
• Culture inoculation:
•: SDA with cycloheximide (actidione), chloramphenicol, and gentamicin or DERMASEL agar.
•: SDA-CG (Sabouraud’s Dextrose Agar with Chloramphenicol & Gentamicin).
• Incubate at 26°C for 4 weeks.
KOH wet mount or Calcofluor stain
Scrapings
Swabs
Differentiates Candida species and other yeasts.
Cornmeal with Tween 80 Agar
Selective for Cryptococcus neoformans (produces brown colonies).
Birdseed Agar (Staib Agar)
Isolates saprophytic and pathogenic fungi.
Brain Heart Infusion (BHI) Agar
Common primary culture medium for fungal isolation
Sabouraud’s Dextrose Agar (SDA)
What can be seen under a Wood’s lamp?
✅ Hair – Certain fungal infections (e.g., Microsporum species) cause fluorescence.
✅ Skin – Some dermatophytes and bacterial infections fluoresce.
✅ Nails – Most fungal nail infections (onychomycosis) do NOT fluoresce.
📝 Answer: Hair, Skin
Kills live organisms and preserves fungal structure.
Blue-stained fungal elements.
Lactophenol Cotton Blue (LPCB)
Clears background debris and enhances fungal visibility.
Fungal elements appear refractile.
Potassium Hydroxide (KOH)
Binds to fungal cell walls, observed under fluorescence.
Blue-white fluorescence under UV light.
Calcofluor White Stain
Stains fungi magenta by binding to polysaccharides.
Fungal structures appear bright magenta.
Periodic Acid-Schiff (PAS)
Stains fungal cell walls black.
Black fungal elements, green background.
Gomori Methenamine Silver (GMS)
