Fungal Culture

Fungi (yeasts and molds) are important causes of human disease, food spoilage, and environmental contamination. Their culture requires specific media and incubation conditions.

Sabouraud dextrose agar (SDA) is the standard medium for fungal isolation. It has a low pH (5.6) that inhibits bacterial growth. Chloramphenicol or gentamicin is often added to suppress residual bacterial contamination. Inhibitory mold agar (IMA) is an alternative for clinical specimens.

Incubation is at 25–30 °C for environmental fungi and 35–37 °C for pathogenic fungi. Plates are held for up to 4 weeks before reporting as negative (molds grow slowly). Yeasts typically grow within 24–48 hours.

Identification is based on:

• Colony morphology: texture (powdery, cottony, velvety), pigment, growth rate, reverse color.
• Microscopic morphology: hyphal type (septate vs. non-septate), conidiophore structure, spore shape and arrangement. The lactophenol cotton blue (LPCB) mount is the standard preparation for visualizing fungal structures from a colony.
• Biochemical tests for yeasts: assimilation of carbon sources (API 20C AUX, VITEK YST), germ tube test (Candida albicans is positive).

Yeast culture: most yeasts grow on standard bacteriological media (blood agar, chocolate agar) as well as SDA. The germ tube test for C. albicans involves incubating a colony in serum at 37 °C for 2–4 hours and checking for hyphal outgrowth under the microscope.

Anaerobic Culture

Anaerobic bacteria (Clostridium, Bacteroides, Fusobacterium, Peptostreptococcus) are killed or inhibited by oxygen. Their culture requires oxygen-free conditions throughout handling and incubation.

Specimen collection: obtain the specimen anaerobically (syringe aspiration, swab in anaerobic transport medium). Avoid exposing the sample to air. Transport to the laboratory within 30 minutes.

Anaerobic media:

• Pre-reduced anaerobically sterilized (PRAS) media: boiled and stored in oxygen-free gas (N₂, CO₂, H₂).
• Brucella blood agar with hemin and vitamin K.
• Phenylethyl alcohol agar: suppresses facultative anaerobes.
• Kanamycin-vancomycin laked blood agar: selective for Bacteroides and other Gram-negative anaerobes.

Anaerobic incubation:

• Anaerobic jar: a sealed container with a gas-generating envelope that produces H₂ and CO₂. Palladium catalyst beads catalyze the reaction of H₂ with residual O₂ to form water. An indicator strip (resazurin) turns colorless when anaerobic conditions are reached.
• Anaerobic chamber: a sealed glove box with an atmosphere of 85% N₂, 10% H₂, 5% CO₂. Palladium catalyst removes trace oxygen. Allows continuous manipulation without exposing cultures to air.
• GasPak system: a self-contained disposable envelope system for small numbers of plates.

Incubation is at 37 °C for at least 48 hours. Anaerobes typically grow more slowly than aerobes.