Fungal Culture - Collection, Incubation, Temperature, Media

Introduction to Fungal Culture

Medically, fungal culture is the method of growing yeasts and molds in the laboratory for research and/or diagnosis of fungal infections. For fungal isolation, cultivation, and maintenance, the most commonly used non-selective media is Sabouraud dextrose agar (SDA).

This method is a gold standard in mycology.

Collection for Fungal Culture

Specimens for fungal culture include blood, CSF, pus, urine, tissue, respiratory samples (sputum, BAL), pleural, pericardial or peritoneal fluid, skin scraping, hair, nail clippings, oral or vaginal samples. For Blood and bone marrow samples, the anticoagulant Sodium Polyanethol Sulfonate (SPS) is added.

After the collection of samples following the sterile technique, it is stored in a sterile container within 2 hours. In case of delay, the specimen must be stored at 4°C.

In the case of solid-state samples, they are processed by centrifugation of softening/liquidisation and spread onto the desired agar medium.

Temperature for Fungal Culture

For the culture of fungi in a laboratory, the optimum temperature of the incubator should be maintained at 25 to 30°C.

In the case of dimorphic fungi, two sets of media must be inoculated and incubated. One set of media must be incubated at 37°C (for yeast form) and another set incubated at room temperature (25°C) for mold form. On the fungal culture media, yeasts show isolated colonies while filamentous fungi mostly have white cottony growth.

For cultures obtained from the respiratory tract, the inoculated media plates must be incubated at 42°C as it prevents the growth of Candida spp. at the same time, promoting the growth of Aspergillus spp.

Incubation time for Fungal Culture

Some species of fungal colonies can be visualized within 24 to 72 hours after incubation. Such fungi include Candida, Aspergillus, Mucor, and Rhizopus.

On the other hand, many fungi can be slow growing and may take up to 2-3 weeks to culture such as Histoplasma spp. Fastidious fungi should be incubated for 8 weeks.

For at least a week, the culture plates must be inspected daily for signs of fungal growth. From the second week, inspection of 3 times a week is recommended.

Since the incubation time for fungal culture and hence diagnosis may take from days to weeks, molecular methods have been used. More than 95% of positive cultures can be identified within seven days by molecular detection.

Growth Media for Fungal Culture

Fungal culture media such as Sabouraud dextrose agar (SDA) can be supplemented with antibiotics such as gentamicin and chloramphenicol to minimize bacterial contamination.

Cycloheximide if added inhibits the growth of saprobic fungi. However, for suspected cases of Cryptococcus spp., Aspergillus spp., and Scytalidium spp., cycloheximide should not be used as these fungi are sensitive to antibiotics.

Special/selective media can also be used for fungal growth and culture. For example, Bird seed agar / Niger seed agar can be used for culture of Cryptococcus neoformans.

Commonly used media for fungal culture

• Sabouraud dextrose agar (SDA)

• Potato dextrose agar (PDA)

• Birdseed Agar / Niger Seed Agar / Staib Agar

• Rice starch Agar

• Cornmeal (tween) Agar

• Potato Flake Agar

• Brain-Heart Infusion (BHI) Agar

• CHROMagar Candia medium

• Mycosel/mycobiotoc Agar

• Czapek-Dox Agar

• Inhibitory Mold Agar (IMA)

• Dermatophyte test medium (DTM)

• Fungal Blood Culture Systems

• Modified Leeming-Notman Agar

• Modified Dixon Agar

• Christensen’s Urea Agar

• Esculin Agar

• Trichophyton Agar (six types of media are present - Trichophyton Agar 1,2,3,4,6,7)