The clinical laboratory has access to a variety of enrichment and selective media for the isolation and identification of pathogenic fungi. The majority of the media mentioned in this article are commercially available in dehydrated or finished form. Recommendations for general media use, enrichment or selective media use, or combinations of the two are outlined in the medical mycology and culture media category of labweeks.
1. Antifungal Susceptibility Testing Media
The Clinical and Laboratory Standards Institute (CLSI) documents M27-A3 and M38-A2 describe procedures for determining the susceptibility of yeast and filamentous fungi to antifungal agents through broth dilution. The recommended medium is an RPMI 1640 medium that does not contain sodium bicarbonate but does contain L-glutamine. To keep the pH of the medium at 7.0, 3-N-morpholinopropanesulfonic acid (MOPS) is added as a buffer. The yeast nitrogen base broth containing 2% glucose can also be used for broth dilution procedures.
2. Assimilation Base for Carbohydrates
Modified yeast nitrogen base is a synthetic basal medium that contains enough nitrogen to support fungi growth during carbohydrate assimilation testing. Plated fungal isolates are grown confluently, and carbohydrate disks are dispensed onto the surface to provide the specific carbohydrates for assimilation testing. After 48 to 72 hours of incubation at 30°C, the plates are examined for growth around each disk. Good growth around a disk indicates carbohydrate assimilation, whereas scant or no growth around a disk indicates no assimilation. All yeasts use glucose (dextrose) as a growth regulator.
The basal media can also be prepared as a broth to which individual carbohydrates are added in separate tubes. The yeast is placed in a series of tubes. The tubes are incubated, and the broth becomes turbid if the yeast can assimilate the carbohydrate. The carbohydrate assimilation pattern is used to identify the yeast.
3. Birdseed Agar
Birdseed agar is a selective and differential enrichment medium used to isolate and identify Cryptococcus neoformans. Guizotia abyssinica ground seeds provide enrichment as well as caffeic acid, which serves as a substrate for the detection of phenol oxidase activity. Because phenol oxidase activity results in the deposition of melanin in yeast cell walls, C. neoformans colonies typically darken to a rich brown on this medium. Other Cryptococcus spp. and yeast colonies remain cream-colored. Bacterial growth can be inhibited by the addition of chloramphenicol.
4. Brain-Heart Infusion Agar
Brain-heart infusion (BHI) agar containing 10% sheep blood is an enrichment agar that can be used to isolate pathogenic yeast and dimorphic fungi (in yeast form) from clinical specimens. The addition of antimicrobial agents, such as chloramphenicol and gentamicin or penicillin and streptomycin, selectively alters the medium by inhibiting bacterial growth.
5. Canavanine-Glycine–Bromothymol Blue Agar
This differential medium distinguishes Cryptococcus neoformans from Cryptococcus gattii. Plates are inoculated and incubated at 30°C for 1 to 5 days. Colonies of C. gattii are cobalt blue, indicating glycine assimilation, whereas colonies of C. Neoformans grow slowly and produce a medium greenish-yellow waste. Glycine is the sole carbon source; assimilation of glycine and subsequent conversion of creatine to ammonia causes an alkaline shift in the pH, resulting in blue colonies. The medium favors Cryptococcus because they are the only yeasts known to have a natural resistance to L-canavanine. Bromothymol blue is added to indicate the shift in pH to 7.0 when the media appears cobalt blue.
6. Chromogenic Agar for Candida
Several manufacturers’ chromogenic agars provide rapid and accurate identification of Candida spp. isolated in clinical laboratories. Based on species-specific enzymes, chromogenic substrates in agar produce a variety of colored products. CHROMagar Candida (BD Diagnostic Systems, Sparks, MD) contains chloramphenicol to inhibit bacteria; this medium is more selective than Sabouraud dextrose agar. C. albicans colonies are yellow-green to blue-green in color, C. tropicalis colonies are dark blue to metallic blue in color, and C. krusei colonies are light mauve to mauve in color. After a 48-hour incubation under aerobic conditions at 35°C, colonies should be examined. Another example is ChromID Candida agar (bioMérieux, Hazelwood, MO).
7. Cornmeal Agar
Cornmeal agar comes in a variety of formulations. Each variant is recommended for cultivation or enhancement of specific fungal characteristics. Cornmeal agar without dextrose (glucose) is recommended for the cultivation of chlamydosporebearing C. albicans, with chlamydospore production boosted by the addition of 1% Tween-80. Cornmeal agar with 1% glucose is not recommended for the growth of chlamydospores. Rather, this formulation promotes more luxuriant growth and increases pigment production, which is used to distinguish Trichophyton rubrum, which produces a red pigment, from Trichophyton mentagrophytes, which does not.
Without cutting into the agar, inoculate each plate with a known C. albicans control and the unknown isolate(s) as single streaks; cover the streak lines with a flame-warmed coverslip. The coverslip creates a low-oxygen environment that promotes chlamydosporulation. For up to a week, the plate should be incubated at room temperature in the dark and examined with a low-power lens of a microscope. Isolates can become positive for chlamydospores (macroconidia) within 48 hours, but they cannot be considered negative until the fifth day of culture.
8. Dermatophyte Test Medium
Dermatophyte test medium is used to isolate dermatophytes from skin samples. Most bacteria are inhibited by chloramphenicol and gentamicin, while saprophytic fungi are inhibited by cylcoheximide. When dermatophytes grow, the medium turns from yellow to red due to the formation of alkaline metabolites. The pH indicator is phenol red. The colonies growing on this medium show the dermatophyte’s microscopic characteristics.
9. Inhibitory Mold Agar
This is an enriched selective medium for cycloheximide-sensitive fungi like Cryptococcus, Histoplasma capsulatum, and mucoraceous fungi. Most bacteria are inhibited by chloramphenicol and gentamicin. Casein, yeast extract, and animal tissue all provide nutrients.
10. Littman Oxgall Agar
Littman oxgall agar is a general purpose medium used to isolate fungi, particularly dermatophytes. It is similar to inhibitory mold agar in that it does not contain cycloheximide. Glucose is added as a carbon source, peptone as a nitrogen source, and oxgall prevents fungal colonies from spreading so that yeasts and molds form distinct colonies and can be isolated in pure culture. To inhibit bacterial growth, crystal violet and streptomycin are added.
11. Modified Potassium Nitrate Assimilation Medium
The potassium nitrate (KNO3) assimilation medium is a differential medium that can be used to test yeast’s ability to assimilate KNO3. The modified medium is a slanted solid medium that contains KNO3, yeast carbon base, Nobel agar, and bromothymol blue. A single colony of the cultured isolate is picked with a sterile applicator stick, inoculated across the entire slant surface, and incubated at 25° to 30° C. The ability to absorb potassium nitrate is indicated by a change in the color of the medium from greenish-yellow to blue or blue-green.
12. Mycosel/Mycobiotic Agar
Mycosel (BD Diagnostic Systems) and Mycobiotic (Remel, Lenexa, KS) are pathogenic fungi, dermatophytes, and systemic pathogens isolation media. Cycloheximide inhibits the growth of saprophytic fungi, whereas chloramphenicol inhibits the growth of bacterial contaminants.
13. Potato Dextrose Agar
The recommended plating medium for the cultivation, enumeration, and identification of yeasts and molds from products and clinical specimens is potato dextrose agar (PDA). The potato infusion promotes fungi growth and sporulation. This medium promotes the production of dermatophyte pigments. It is frequently used in slide culture to visualize the microscopic morphology of fungi, including mycelia and reproductive structures.
14. Potato Flake Agar
To induce sporulation in fungi, potato flake agar is used. It is less difficult to make and more stable than potato dextrose agar. The nutrients are provided by potato flakes and dextrose. The pH is adjusted to approximately 5.6 to promote fungi growth while slightly inhibiting bacterial growth. To make the medium more selective for fungi, particularly dermatophytes, a modified version includes cycloheximide and chloramphenicol.
15. Rice Extract Agar
Rice extract agar without additional dextrose is effective in the cultivation of Candida albicans, increasing chlamydospore production. Rice extract agar with 2% dextrose has been shown to increase pigment production in T. rubrum, allowing it to be distinguished from T. mentagrophytes.
The medium should be lightly inoculated on the surface of the in a manner similar to that described for cornmeal agar inoculation. To stimulate chlamydospore production, cover the streaks with a flamewarmed coverslip. All cultures should be incubated for 18 to 72 hours at room temperature.
16. Rice Grains Medium
Microsporum audouinii can be distinguished from other dermatophytes using rice grains medium, particularly Microsporum canis. M. audouinii does not grow well on this medium and discolors it. Other dermatophytes and most other fungi grow and sporulate well on this medium, with no medium discoloration. To avoid confusion when distinguishing between discoloration and actual growth, sterile rice grains should be spot-inoculated.
17. Sabouraud-Brain-Heart Infusion Agar
Sabouraud-brain-heart infusion agar (SABHIA) is a general purpose fungi isolation medium. It is made from Sabouraud dextrose and BHI ingredients. This medium is especially beneficial for isolating dimorphic fungi from clinical specimens. The addition of blood promotes the isolation of dimorphic fungi and the transition to the yeast stage. To increase the selectivity of the medium for fungi, antimicrobials such as chloramphenicol, cycloheximide, streptomycin, and penicillin can be added.