Fungal Media "demystified"

CULTURE MEDIA FOR FUNGI

Clinical specimens are processed promptly and plated on isolation media to recover fungi that may be causing disease. Media and incubation temperatures are selected to allow for the growth of pathogenic and opportunistic yeasts and fungi.

NB. If Histoplasma, Coccidioides, Paracoccidioides, Blastomyces or Penicillium marneffei are suspected, Category 3 Containment is required.

ISOLATION MEDIA

  • Media must be carefully selected based on specimen type and fungal suspected agents.
  • Media is dispensed into containers such as screw cap tubes or Petri dishes.
  • All inoculated media should be read every 2 days following incubation and twice weekly thereafter.
  • Plates must be opened only within a biological safety cabinet to prevent contamination of the plate and exposure of personnel to potentially dangerous fungi.
  • If the specimen is from a contaminated site, it is important to include media that contain inhibitory substances such as chloramphenicol, gentamycin, or cycloheximide.
  • Chloramphenicol or gentamycin will inhibit most bacterial contaminants, while cycloheximide inhibits most saprobic moulds.
  • It is important to remember that cycloheximide may also inhibit opportunistic fungi such as some species of Aspergillus, Fusarium, Scopulariopsis, Pseudallescheria, zygomycetes, some dematiacious fungi, and yeasts such as Cryptococcus neoformans and some Candida species. Antibacterial agents may inhibit the growth of aerobic actinomycetes like Nocardia sp.
  • It is important to use media with and without inhibitory agents. Specimens from normally sterile sites can be inoculated to media without inhibitory substances.

Sabouraud Dextrose Agar (SAB)

  • SAB agar (Emmons' modification contains 2% glucose and is slightly acidic (pH 6.5). It is the standard medium for recovery and maintenance of a wide variety of fungi commonly isolated in the clinical laboratory. The original SAB formulation specifies 4% glucose. Emmons' modification with less glucose is preferred as an isolation medium because some isolates, notably Blastomyces dermatitidis may not be recovered using the original Sabouraud formulation.
  • SAB + Chloramphenicol + Cycloheximide - Mycosel (BBL) and Mycobiotic (Difco) agars are commercially prepared media containing SAB agar, 1% glucose, chloramphenicol, and cycloheximide. These media are used for the selective recovery of dimorphic fungi and dermatophytes.

Brain-Heart Infusion Agar (BHI)

  • BHI is an enriched medium that enhances the recovery of Cryptococcus neoformans from sterile specimens such as CSF. BHI is also used in yeast-mould conversions for Sporothrix and Paracoccidioides.
  • Brain-Heart Infusion Agar + Gentamycin + Chloramphenicol (16 μg/ml) + 10% sheep's blood. BHI + GC + 10% bl is an enriched medium useful for the recovery of fungi such as C. neoformans from contaminated specimens.
  • Niger (bird) seed agar.Niger seed agar is used for the isolation of C. neoformans. Colonies turn brown on this medium due to the production of melanin. Can also be used to  discriminate between C. albicans and C. dubliniensis. C. albicans produces only yeast cells on this medium after 24 h at 37°C, while C. dubliniensis produces extensive hyphal and pseudohyphal growth that is easily observed (Diagn Microbiol Infect Dis. 2003 May;46(1):13-7).

Inhibitory Mould Agar (IMA)

  • IMA is an enriched medium with inorganic salts, chloramphenicol, and gentamycin. It is useful for inhibiting bacteria.

Blood Culture Media

  • Two plated media are commonly used in conjunction with Isolator Lysis centrifugation:
    • Inhibitory Mould Agar with chloramphenicol and gentamycin for the isolation of fungi and the inhibition of bacteria.
    • Brain-Heart Infusion Agar for fungi and bacteria, which may be substituted for Chocolate Agar.

Brain-Heart Infusion Broth

  • BHI broth with penicillin is added to the normal battery when zygomycetes are suspected. These fungi can be very difficult to recover. The use of broth provides optimal  medium/specimen contact. The aseptic addition of sterile penicillin discs will inhibit bacteria. Malt extract agar is an effective alternative to broth media for the isolation of zygomycetes.

Sterile Bread

  • Sterile bread without preservatives is recommended for the recovery of zygomycetes from clinical specimens. Bread is often superior to other media for recovering this group of opportunistic pathogens. A piece of bread is sterilized in a humidified Petri dish. Specimens from non-contaminated sites can be directly inoculated.
  • Contaminated specimens should be treated with antibacterial agents before inoculation. Zygomycetes will grow rapidly, often filling the entire Petri dish within a few days.

Malt Extract Agar

  • Malt agar is a useful alternative to bread for recovery of zygomycetes and is excellent for environmental cultures.

Yeast Extract-Phosphate Medium (YEP)

  • YEP medium was developed for the enhanced recovery of Blastomyces dermatitidis and Histoplasma capsulatum from contaminated specimens. The incorporation of chloramphenicol inhibits bacteria and the addition of a drop of concentrated ammonium hydroxide (approximately 58%) inhibits bacteria and yeasts.

Dermatophyte Test Medium (DTM)

  • DTM is used to recover dermatophytes from heavily contaminated clinical specimens and to presumptively indicate the presence of a dermatophyte. Dermatophytes, as well as a few other fungi and bacteria turn the medium from pink to red.

AMH.