Mycology: Culture Media and Specimen Handling

Culture Media, Stains, and Biochemicals in Mycology

• Mycology is the study of fungi
• Culture medias, stains, and biochemical tests are used to identify fungi.

Specimen Collection, Handling, and Processing

• Skin: Cleaned with 70% alcohol to remove dirt and surface saprophytes
• Nails: Cleaned similarly to skin
• Hair: Obtained by plucking from the infected area of the scalp
• Body fluid: Collected using a normal sterile procedure
• Skin, Hair, and Nails: Examined using KOH (Potassium Hydroxide)
• Cerebrospinal fluid (CSF): Centrifuged, and the sediment examined microscopically
• Pleural fluid, sputum, and bronchial aspiration: Fresh specimens; refrigerated if delay is expected up to 2 hours.
• Gastric washings: Handled similarly to pleural fluid
• Genitourinary specimens: First morning urine sample
• Bone marrow: Typically inoculated directly into brain-heart infusion (BHI) broth
• Wound abscess or drainage: Cultured anaerobically
• Tissue specimens: Examined for pus, caseous material, or granules

Direct Examination of Specimens

• Direct microscopy: Required for any material sent for fungal culture
• Look for spores, hyphae, mycelial elements, budding yeast, and mycotic granules. (Advanced rapid, simple, and easy technique with minimal equipment).
• Wet mount prep: Good for yeast examination
• KOH prep: Used for skin scrapings, nails, sputum, and vaginal specimens. KOH clears tissue and mucus and makes fungal elements visible (10-40%).

Stains Used in Mycology

• Lactophenol Cotton Blue (LPCB): Popular for quick evaluation of fungal structures. Stains chitin in the cell wall of fungi
• Phenol: Disinfectant
• Lactic acid: Preservative for morphology
• Glycerol: Prevents drying
• Cotton blue: Stain.
• Periodic Acid-Schiff (PAS): Stains certain polysaccharides in the cell wall of fungi. Staining appears pink-red with blue nuclei
• Gomori Methenamine Silver Stain: Highlights fungi in black due to silver precipitates. The background is light green
• Gridly stain: Hyphae and yeasts stain dark blue (deep tissue)

Other Stains

• Fluorescent Antibody stain: Simple, sensitive, and specific
• Papanicolaou stain: For differentiating dimorphic fungi (sputum)
• Gram stain: Most fungal samples will appear gram-positive
• Modified-Acid Fast Stain: Useful in differentiating acid-fast Nocardia from other samples
• Giemsa stain: Used for blood and bone marrow
• India Ink: Demonstrates the capsule of Cryptococcus neoformans in CSF

Fungal Culturing

• Plate media: Often preferred over tube media
• Tube media: Adequate surface area, less chance of spore release or dehydration; easier to store
• Plate media: Saprotrophic fungi grow faster
• Acidic pH: May inhibit significant pathogenic fungi
• Incubation: Aerobic conditions typical; anaerobic conditions for Actinomycetes
• Incubation in 37°C if dimorphic fungi suspected. Room temperature otherwise.
• Typically takes 10 days to 1 month to observe growth

Media Used in Medical Mycology

• Basal Media: Sabouraud Dextrose Agar (SDA), SDA with antibiotics, Sterile Bread
• Nutritionally Deficient Media: Corn Meal Agar, Rice Starch Agar
• Enriched Media: Brain-Heart Infusion Agar, Cysteine-Heart and Hemoglobin Agar, Biphasic Medium, Blood Agar, Lowenstein-Jensen Medium, Dermatophyte Test Medium(DTM), Dermatophyte Identification Medium, Czapek-Dox Agar, PYG Agar Medium, Malt extract Agar

SDA with Antibiotics (CSDA)

• Used for isolating pathogenic fungi
• Cycloheximide (500 mg), Chloramphenicol (50 mg), and optionally Gentamicin (20 mg) prevent contaminating bacteria and saprotrophic fungi
• Cycloheximide: Dissolved in 2 ml acetone
• Chloramphenicol / Gentamicin: Dissolved in 10 ml ethanol and added to boiling medium

Neutral SDA & Sterile Bread

• Neutral SDA: Lower dextrose concentrations (20g/L instead of 40g/L), lower glucose, and neutral pH
• Sterile Bread: Inoculation without preservatives, humidified in Petri dish; used for specimen sites contaminated with bacteria

Nutritionally Deficient Media (Corn Meal Agar, Rice Starch Agar)

• Corn Meal Agar: 60°C for one hour heating of corn meal and water. Filter, add water and sugar, and autoclave. Used for observing morphological details of yeasts like Candida albicans and Candida dubliniensis .
• Rice Starch Agar: Add cream of rice to boiling water, filter, and add water to required volume. Used to produce chlamydospore of Candida albicans and Candida dubliniensis.

Enriched and Selected Media (Brain-Heart Infusion Agar)

• Used to grow fastidious fungi like Histoplasma capsulatum and Blastomyces dermatidis
• Useful for isolation and converting mycelium to yeast form and vice-versa.

Cysteine-Heart and Hemoglobin Agar

• Alternative to Brain-Heart Infusion Agar
• Contains dehydrated cysteine, hemoglobin, distilled water, and antibiotics

Biphasic Medium

• Used for isolating fungi in blood cultures. Includes a blood agar and broth.

Blood Agar

• Routinely used in bacteriology, also applicable for growing fungi. Contains sheep blood, distilled water, and agar. Applicable for isolating Histoplasma capsulatum and Blastomyces dermatidis

Lowenstein-Jensen Medium

• Isolates Madurella, and Nocardia species
• Contains hen's egg, salt solution, asparagine, and malachite green for bacteria inhibition.

Dermatophyte Test Medium

• Used for identifying dermatophytes (hair, nail, and skin fungi)
• Most produce a red colour that increases in pH
• Contains Phyton, Dextrose, phenol red solution, HCL, Acitidione, Gentamicin, agar, and distilled water. Incubated at 25 degrees cellcius
• Limited use due to failure to inhibit numerous saprotrophic fungi.

Dermatophyte Identification Medium

• Used for presumptive identification of dermatophytes
• Contains Dextrose, Neopeptone, Cycliheximide, Penicillin, Streptomycin, and Bromocresol purple. Incubated at 37°C.
• Greenish blue to purple colour change indicates growth of dermatophyte within 1–2 days

Malt Extract Agar

• A substitute for SDA (Sabouraud Dextrose Agar).
• Can be acidified using hydrochloric acid to reduce pH (pH < 4)
• Contains Malt extract, peptone, glucose, agar, and distilled water

Other Media

• Includes various agar mediums for fungi cultivation, like Bird Seed Agar, Sunflower Seed Agar, Oatmeal Agar, DRBC Agar, Dextrose Agar, and Czapek-Dox Agar.
• PYG and Potato Agars are also listed

Media Used for Biochemical Tests (Tetrazolium Reduction Medium)

• Used to differentiate Candida species
• Includes Peptone, Glucose, Beef Extract, Tetrazolium, Neomycin, and Distilled Water

Carbohydrate Fermentation Media

• Contains peptone, NaCl, Andrade indicator, and sugars like dextrose, lactose, maltose, and sucrose.
• Useful for determining acid and gas production by fungi
• Incubated for 1-2 days at 37°C

Urease Medium

• Used to detect urease enzyme production in yeast
• Production of ammonia raises pH and changes the colour from amber to pink red due to phenol as the indicator. Differentiate dermatophytic fungus
• Rapid Urease Test: Swab is used for colour change determination.

Additional Information

• Various media specifications are provided with ingredients and pH values for many of the listed media.