Czeum Project Culturing protocols

Culturing protocols

Chytrid and blastoclad fungi in culture are primarily saprobic heterotrophs, with notable pathogenic exceptions on algae, plants, insects, other fungi, and, in the case of Batrachochytrium, amphibians. Consequently, many types of culture media are necessary to maintain this diversity. The recommended medium for each culture is recorded in the database. Here we discuss maintenance by sterile transfer and how to avoid contamination, list culture media recommended by the CZEUM for culturing zoosporic eufungi, and describe our cryopreservation protocol.
Unless otherwise requested, we provide CZEUM cultures on agar medium plates without antibiotics. Though zoosporic eufungi are by nature aquatic organisms, we recommend that cultures on agar media be kept inverted and void of free-flowing water for routine maintenance. Without excessive drying, the surface of the agar is an acceptable environment for limited zoospore dispersal. Excessive moisture can precipitate and permeate into seams between the plate and lid, potentially making contact with the outside environment and bringing in contaminants. To maintain sterility, always transfer zoosporic eufungi in a laminar flow cabinet. To inoculate a new agar medium plate, remove an inoculation plug from the culture plate using a sterile scalpel, place the plug inverted on a new plate such that the thalli of the zoosporic fungi are in contact with the fresh agar surface, and push the agar plug around the surface of the plate to remove some of the thalli from the plug. Pushing the agar plug along the surface of the new plate may also physically displace any contaminants that were also transferred and may also increase the area on the new plate that is covered by the fungal culture. Monitor growth of the fungus of the closed agar plate with either a stereo dissecting microscope or invert the plate and observe the culture through the agar with a compound microscope at low magnification.
In order to stimulate zoospore production 1-2 ml of sterile distilled water can be added to a plate once the first zoospores are observed. After 30 minutes of saturation, cultures often will release copious zoospores into the water which can be harvested and used.
Unless otherwise requested, we provide CZEUM cultures on agar medium plates without antibiotics. If cultures arrive with minor contamination, observe the plate with a microscope to determine if the contamination is restricted to a certain area. If this is the case, transfer a plug of the non-contaminated agar with zoosporic eufungi to a fresh agar medium plate with antibiotics; we recommend 200mg/L penicillin G and 300mg/L streptomycin mixed into the medium post- autoclaving. Place the inoculation plug such that the thalli of the zoosporic fungi are in contact with the fresh agar surface. Pushing the agar plug along the surface of the new plate may also physically displace any contaminants that were also transferred and may also increase the area on the new plate that is covered by the fungal culture. Monitor growth of the fungus, repeating the above process if necessary, until contamination is no longer observed. At this time, it should be safe, but not required, to transfer the fungus to media without antibiotics.
Autoclave all culture media before preparing agar plates or liquid media. For liquid media, remove agar from recipes below.

  1. 1% Tryptone (1%T)


  2. 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.

  3. Cd; Longcore and Simmons 2012


  4. 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.

  5. C-T


  6. 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.

  7. dss


  8. 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.

  9. E/4 (Emerson’s ¼ strength)

  10. Emerson’s medium is YPSS. See YPSS/4 for recipe.

  11. 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

  12. 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.

  13. mPmTG (modified PmTG, or m); Longcore 1992


  14. 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.

  15. PmTG (P); Barr 1986


  16. 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.

  17. PmT; Simmons et al 2009


  18. 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.

  19. 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)

      • 20 grams Agar
      • 0.25 gram K2HPO4.3H20 (dibasic)
      • 0.125 gram MgSO4.7H2O
      • 3.75 grams Soluble Starch
      • 1 gram Yeast Extract
      • 1 liter Distilled Water

    3. YpSs/8 (Y/8)

      • 20 grams Agar
      • 0.125 gram K2HPO4.3H20 (dibasic)
      • 0.0625 gram MgSO4.7H2O
      • 1.875 grams Soluble Starch
      • 0.5 grams Yeast Extract
      • 1 liter Distilled Water

    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.
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.
Barr DJS. 1986. Allochytridium expandens rediscovered: morphology, physiology and zoospore ultrastructure. Mycologia. 78:439–448. http://dx.doi.org/10.2307/3793048

Boyle DG, Hyatt AD, Daszak P, Berger L, Longcore JE, Porter D, Hengstberger SG, Olsen V. 2003. Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes. Diseases of Aquatic Organisms 56:59–64. http://dx.doi.org/10.3354/dao056059 Emerson R. 1958. Mycological organization. Mycologia 50:589 –621. http://dx.doi.org/10.2307/3756168

Longcore JE. 1992. Morphology, occurrence and zoospore ultrastructure of Podochytrium dentatum sp. nov. (Chytridiales). Mycologia 84:183–192. http://dx.doi.org/10.2307/3760849

Longcore JE, Simmons DR. 2012. The Polychytriales ord. nov. contains chitinophilic members of the rhizophlyctoid alliance. Mycologia 104:276–294. http://dx.doi.org/10.3852/11-193

Murray CL, Lovett JS. 1966. Nutritional requirements of the chytrid Karlingia asterocysta, an obligate chitinophile. American Journal of Botany 53: 469–476. http://dx.doi.org/10.2307/2440345

Simmons DR, James TY, Meyer AF, Longcore JE. 2009. Lobulomycetales, a new order in the Chytridiomycota. Mycological Research 113:450–460. http://dx.doi.org/10.1016/j.mycres.2008.11.019