Media Recipes

Autoclave all culture media before preparing agar plates or liquid media. For liquid media, remove agar from recipes below.

1% Tryptone (1%T)

  • 10 grams Agar
  • 10 grams Tryptone
  • 1 liter Distilled Water

CZEUM uses this medium exclusively for isolates of Batrachochytrium dendrobatidis (Bd). Though Longcore initially isolated Bd using TGhL agar (Longcore et al. 1999), she switched to using 1%T shortly thereafter as a simpler, effective alternative.

Cd; Longcore and Simmons 2012

  • 10 grams Agar
  • 4 grams Cellobiose
  • 0.4 grams Peptonized Milk
  • 4 grams Soluble Starch
  • 0.4 grams Tryptone
  • 1 liter Distilled Water

This medium was developed by Longcore as a consolidation of two culture media, C-T and dss. Containing both cellobiose and soluble starch, this medium may be preferable when isolating zoosporic eufungi from aquatic plant detritus.

C-T

  • 10 grams Agar
  • 4 grams Cellobiose
  • 0.4 gram Tryptone
  • 1 liter Distilled Water

This medium was previously used by Longcore for isolating zoosporic eufungi from aquatic plant detritus. However, Longcore consolidated this and another medium to make Cd medium (Longcore and Simmons 2012). Should all of the ingredients be available to you, the CZEUM staff recommends using Cd medium.

dss

  • 10 grams Agar
  • 0.2 gram Peptonized Milk
  • 4 grams Soluble Starch
  • 0.2 gram Tryptone
  • 1 Liter Distilled Water

This medium was previously used by Longcore for isolating zoosporic eufungi from aquatic plant detritus. However, Longcore consolidated this and another medium to make Cd medium (Longcore and Simmons 2012). Should all of the ingredients be available to you, the CZEUM staff recommends using Cd medium.

E/4 (Emerson’s ¼ strength)
Emerson’s medium is YPSS. See YPSS/4 for recipe.

LB

  1. Boil 1 package of Frozen Lima Beans for 0.5 hr in 1 liter of Sterile Distilled Water
  2. Filter the solution through cheese cloth to remove lima beans
  3. Add Distilled Water to reach final volume of 1 liter
  4. Add 10 grams Agar

M&L; Murray and Lovett 1966

  1. Modified Machlis’ Solution
    • 4 grams NH4NO3 (Final Conc: 5 X 10-3 M)
    • 0.72 gram CaCl2 (Final Conc: 5 X 10-4 M)
    • 0.6 gram MgSO4 (Final Conc: 5 X 10-4 M)
    • 13.4 grams Na2HPO4 (Final Conc: 5 X 10-3 M)
    • 1 Liter Distilled Water
  2. M&L
    • 14 grams Agar
    • 0.5 gram Asparagine
    • 1.2 grams Dextrose
    • 0.6 gram N-Acetyl Glucosamine
    • 2 grams Tryptone
    • 50 mL Modified Machlis’ Solution
    • 1 liter Distilled Water

This medium was originally described for the isolation and study of an obligate chitinophilic chytrid. Longcore and Simmons (2012) found this medium to be useful for members of the Polychytriales and other chitinophilic zoosporic eufungi that were more inclined to grow on chitin than agar medium.

mPmTG (modified PmTG, or m); Longcore 1992

  • 10 grams Agar
  • 2 grams Dextrose
  • 0.4 gram Peptonized Milk
  • 0.4 gram Tryptone
  • 1 liter Distilled Water

This medium contains the same nutrients as PmTG, but at 40% strength. This dilute medium provides a suitable substrate for most chytrids while discouraging contaminants with higher nutrient requirements. Anecdotally, most non-pathogenic cultures in the CZEUM will grow on either this medium or PmTG.

PmTG (P); Barr 1986

  • 10 grams Agar
  • 5 grams Dextrose
  • 1 gram Peptonized Milk
  • 1 gram Tryptone
  • 1 liter Distilled Water

This medium is the default chytrid culturing medium of the JEL and UACCC, and can be used across the Blastocladiomycota and several orders of the Chytridiomycota, especially for saprobic species. Anecdotally, most non-pathogenic cultures in the CZEUM will grow on either this medium or mPmTG.

PmT; Simmons et al 2009

  • 10 grams Agar
  • 1 gram Peptonized Milk
  • 1 gram Tryptone
  • 1 liter Distilled Water

This medium contains the same nutrients as PmTG, with the exclusion of glucose as the primary carbon source. This medium has proven useful for the isolation and maintenance of members of the Lobulomycetales (Phylum Chytridiomycota) and chytrid fungi parasites of green algae.

YpSs (Y); Emerson 1958

  1. YpSs (Y)
    • 20 grams Agar
    • 1 gram K2HPO4.3H20 (dibasic)
    • 0.5 gram MgSO4.7H2O
    • 15 grams Soluble Starch
    • 4 grams Yeast Extract
    • 1 liter Distilled Water
  2. YpSs/4 (Y/4)
  3. YpSs/8 (Y/8)

Developed by Ralph Emerson (1958), YpSs has been used for many zoosporic eufungi cultured for classroom observations (e.g., Chytridiomycetales, Rhizophydiales, Spizellomycetales). Today, the CZEUM uses YpSs primary for the culturing of Allomyces species (Phylum Blastocladiomycota). Less concentrated recipes (i.e., YpSs/4, YpSs/8) can be used to slow the metabolism of Allomyces or make species susceptible to infection by pathogens (i.e., Rozella (Phylum Cryptomycota)) when the host and pathogen are dual members of a pure culture.

Cryopreservation

All chytrid and blastoclad fungi in the CZEUM are kept in a cryopreserved inert state until a culture request is made for revitalization. Cryopreservation and revitalization methods of the CZEUM follow the methods described by Boyle et al. (2003), and are briefly discussed here. Further information can be obtained by contacting CZEUM staff.

  1. Cultures are grown in their preferred liquid medium for 1–2 weeks, sufficient time to enter, but not exceed, the culture’s exponential growth phase. Testing of each individual culture may be necessary to determine the optimal time for cryopreservation. The key factor is that many young and developed thalli are present; deteriorated thalli and active zoospores will not produce a viable culture after cryopreservation.
  2. Cultures are centrifuged at 4000 RPM for 20 minutes in a refrigerated centrifuge set to 4 °C.
  3. To maintain sterility, always transfer zoosporic eufungi in a laminar flow cabinet. The supernatant resulting from centrifugation is removed and reserved for sterilization by autoclaving. The pellet of fungal thalli is resuspended in the cryopreservation medium below.
    1. Cryopreservation medium
      • 80% volume Culture’s preferred sterilized liquid medium
      • 10% volume Dimethyl sulfoxide (DMSO)
      • 10% volume Fetal Bovine Serum (FBS)
  4. The resuspended thalli in cryopreservation medium are distributed into cryopreservation vials and placed in an ultralow freezer in a isopropanol-buffered cryopreservation container.
  5. Once frozen, cryopreservation vials are transferred to dry storage, liquid nitrogen-cooled freezers for long-term archiving.
  6. To revitalize a culture, a cryopreservation vial is removed from the freezer and placed in a 43 °C water bath for 2 minutes. In sterile conditions, the culture in the liquid cryopreservation medium is then aspirated and transferred to a new agar medium plate of the culture’s preferred medium. An equal volume of sterile distilled water is added to the plate to dilute the cryopreservation medium. The plate is sealed and placed in suitable conditions until growth is observed, ranging from 24 hours to several days. At that time, an inoculation plug is transferred to new agar medium, as described above in A. Sterility and Transfer.