Week 1 Lecture 2 Laboratory Diagnosis of Fungal Diseases Flashcards
(33 cards)
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
