Diagnostic Mycology Lab Techniques

Diagnostic Mycology

• Mycology is the study of fungi.
• Specimen transport and storage needs to be done with caution, treating all samples as potentially hazardous materials.

Transport and Storage

• Specimens should be transported rapidly, or refrigerated at 4°C if transport is delayed.
• Do not freeze specimens.
• If the specimen is tissue, add sterile NSS.
• Refrigeration is not a substitute for timely processing.

Transport

• Aseptic conditions are necessary.
• The specimen should be correctly packaged—right side up.
• Sterile containers are required; using double layers (like ziplock bags) and leak-proof containers (like screw caps).
• A cool box is recommended for transport, keeping the specimen between 2°C to 8°C

General Recommendations

• Disinfect bench tops daily.
• Avoid keeping potted plants.
• Don't allow dust to accumulate.
• Examine fungi cultures in a biosafety hood.
• Autoclave all cultures before discarding.

Fungal Identification

• Direct Examination (visual inspection) is not a common method in the Philippines for fungi.
• Examination under microscopic lenses is required

Direct Examination: Microscopic Examination

• Examine the relevant specimen to isolate the fungi.
• Use wet mount with or without dyes.
• Use potassium hydroxide (KOH), optionally with Parker Super Quink blue-black ink.
• The preparation must be examined under a microscope.

KOH (with or without ink)

• Protein, fats, and polysaccharides in the specimen are soluble with KOH, improving tissue clarity and revealing fungal elements.
• Fungal cell walls, composed of glucans and chitins, are relatively resistant to KOH, enabling visualization of these structures.
• Gentle heating enhances the reaction for easier viewing.

KOH with visible hyphae

• Images (pictures) demonstrate fungal hyphae after KOH preparation.

KOH wet mount hair

• Images (pictures) demonstrate hair sample observed under a microscope after a KOH wet mount.

Calcofluor White

• A dye used in textile and paper industries to whiten, and binds cellulose and chitin, fluorescing when exposed to UV and short-wavelength visible light; necessitating a fluorescent microscope

Alcian Blue

• Identifies C. neoformans in cerebrospinal fluid (CSF).

Acid Fast

• Identifies Mycobacteria and Nocardia.

Giemsa

• Examines bone marrow and blood smears

Gram Stain

• Used in examining bacteria and fungi samples.

India Ink

• Detects C. neoformans in CSF, specifically observing a capsule surrounding the fungi.

Methylene Blue

• Used to detect fungi within skin scrapings.

Methenamine Silver

• Detects fungi within histologic (biopsy) sample sections.

Papanicolaou

• Examines bodily secretions to identify malignant cells

Periodic Acid Schiff

• Detects fungi (and glycogen).

Wright's Stain

• Examines bone marrow or Peripheral blood smear (PBS) samples

Problems with Direct Examination

• Negative results do not necessarily rule out a fungal infection.
• False positive results may occur (e.g., fat droplets mimicking yeast; collagen fibers resembling Nocardia filaments).
• Equivocal results need review by multiple readers.
• Cultures are essential to confirm the presence of fungi, even when initial tests are negative

Fungal Culture

• A sufficient amount of the specimen is necessary.
• 1-2 ml is enough to inoculate media.
• Fluids should be concentrated through centrifugation or filtration.
• Temperature is important; room temperature (RT) of 25°C or 37°C incubation will encourage the growth of fungi/yeasts.
• Encouraging dimorphic fungi to produce yeast forms through 37°C incubation will increase the chances of visualization and detection

Time of Culturing Fungi

• Dermatophytes (skin infections): at least 2 weeks of incubation.
• Opportunistic infections: less than a week of incubation.
• Systemic infections requiring 4-6 weeks (and up to 12 weeks for H. capsulatum).
• Prevent dehydration of cultures by maintaining a thick agar volume inside a plastic bag

Fungal Culture Media

• Primary recovery media are used to cultivate fungi, and different media are suitable depending on the type of infection being examined
• Differential test media are used in confirming growth of specific types of fungi.

Primary Recovery Media

• BHIA
• BHI with antibiotics
• BHI blood culture
• Inhibitory mold agar
• Mycosel or mycobiotic agar,SABHI.
• Potato flake agar
• Yeast extract PO4 agar

Primary Recovery Media: Further details

• Brain Heart Infusion Agar (BHI)
• Primary recovery of saprobic & pathogenic fungi.
• BHI with antibiotics: recovery of fungi exclusive of dermatophytes,
• BHI biphasic (in b.c. bottles): recovery of fungi from blood.

BHI for Blood Culture

• BHI is suitable for blood culture

SABHI

• Image (picture) of a positive SABHI culture.

BHIA

• Image (picture) of Histoplasma capsulatum growth in BHIA medium.

Dermatophyte Test Medium

• Screening medium for dermatophytes
• Inhibitory Mold Agar: recovery of exclusive fungi from dermatophytes.
• Potato Flake Agar for saprobic and pathogenic fungi

Mycosel or Mycobiotic Agar

• Primary recovery medium for dermatophytes.
• Cultivates saprobic and pathogenic fungi.
• Yeast Extract PO4 Agar for the recovery of fungi exclusive from dermatophytes

Mycobiotic Agar

• Picture/image of a fungal growth on a mycobiotic medium, showing a reverse/upside-down color.

SABHI

• Image (picture) of positive Sabouraud dextrose agar (SABHI) culture for C. albicans, and S. cerevisiae (a type of yeast).

Yeast Extract Agar

• Contains images/pictures of yeasts on agar.

Differential Test Media (additional types and uses)

• Ascopore Agar
• Corn Meal Agar with Tween 80 and Tryptan blue (for detecting C. albicans based on chlamydospore production)
• Cottonseed conversion Agar (for the conversion of dimorphic Fungi)
• Czapek's Agar (identifying Aspergillus sp.),
• Niger seed Agar for C. neoformans
• Nitrate Reduction Medium
• Potato Dextrose Agar
• Rice Medium for M. audounii
• Trichophyton Agar
• Urea Agar
• Yeast Fermentation Broth
• Yeast Nitrogen Base agar (carbohydrate assimilation).

Identification of Culture

• Morphology
• Macroscopic & microscopic fungal examination
• Physiology (urea utilization, thiamine requirements).
• Multiple Test System (e.g., API AUX System).
• Biotech - nuclear probes

Fungal Cultures (Examination)

• Microscopic examination, using lactophenol cotton blue stain

Specimen Types

• Abscess (aspirated aseptically by physician, with sterile tubes)
• Blood (aseptic collection, minimum of 5 ml added to growth medium to get the appropriate ratio)
• CSF and other body fluids (aseptic collection, lumbar puncture, process immediately, avoid refrigeration)
• Hair (examine for fluorescence under Wood's lamp, extract scalp scales using sterile tools, place on slides)
• Nail clippings/shavings (cleaned with 70% alcohol; scrape 4-5 times; clippings from proximal to distal are preferable; debris underneath nail plate should be collected; use a 1mm curette to collect debris and immerse sample overnight in KOH (10-30%), for easier KOH digestion; placed between 2 slides, stored in envelope/petri dish; store at room temperature)
• Skin scrapings (scrape the active edge, collect exudate, placed between 2 slides and stored in an envelope/petri dish, stored at room temperature)
• Sputum (1st morning sample; collected using a wide-mouth container after brushing & mouthwash, store at 4ºC storage/transport)
• Stool (40% of healthy and 75% of compromised patients with yeasts in GIT)
• Urine (sterile containers; sent immediately; 24-hour urine unacceptable; from catheter bag unacceptable)
• Vaginal secretions (20% of healthy females have yeast as normal flora; vaginal candidiasis is better diagnosed by clinical characteristics and direct examination)

Antifungal Susceptibility Testing

• M27-A3 for yeast testing,
• M38-A2 for mold testing
• M44-A2 for disk diffusion to identify yeasts.
• M51-A for disk diffusion to identify molds.

Explore direct microscopic examinations, staining methods, and specimen collection in a diagnostic mycology lab. Identify fungal elements like hyphae and spores. Learn about common fungal pathogens and proper specimen handling techniques for accurate diagnosis.