I. Objectives: To observe the sexual spores (zygospores) and asexual spores (ballistosporic conidia, capilloconidia, endospores) formed by Basidiobolus ranarum in culture.

II. Materials:

• DAVIS strain of Basidioblus ranarum Eidam (Groff et al., 1991).
• Sterile disposable petri dishes (100 mm) containing CM+ agar
• 17 g Difco corn meal agar
• 2 g glucose
• 1 g yeast extract
• 1 L distilled water and then autoclaved for 45 min at 15 psi)
• sterile Pasteur pipets and pipet bulbs
• sterile distilled water (SDW)
• rubber policeman
• bent glass rod
• alchohol for dipping implements before flaming
• bunsen burner
• compound light microscope
• microscope slides
• cover slips
• lacto-phenol/cotton blue solution (dissolve 2.5 mg aniline blue in 5 ml distilled water; add 5 g lactic acid, 5 g phenol, and 7.5 ml glycerin)

III. Procedure:

• A. Pipet 1 ml SDW onto confluent Petri dish culture of B. ranarum, DAVIS strain.
• B. Move SDW around surface of culture with pre-sterilized rubber policeman.
• C. Collect SDW now containing masses of conidia.
• D. Transfer conidia in SDW to new Petri dish containing CM+.
• E. Spread conidial suspension over surface of CM+ with pre-sterilized bent glass rod.
• F. Incubate at room temperature under normal day-night light system cycle.
• G. Timing:
  1. Hour 0: inoculate plates as described above.
  2. Hour 8-10: only mycelial growth will be visible on plate with a compound microscope (turn the plate upside down, leaving the cover on) and observe the fungal growth.
  3. Hour 32-40: some conidia will be visible at the tips of forming conidiophores (Fig. 1).
  4. Hour 44: Conidia and some capilloconidia (Fig. 2) can be observed adherent to the lid of the Petri dish. Germination by repetition may be observed (Fig. 3).
  5. Hour 52: Zygospores (Fig. 4) are visible if the mycelium from the agar surface is put on a glass slide with lacto-phenol/cotton blue and heated slightly before coverslipping. After 24 h at room temperature, the coverslips can be ringed with nail polish to produce a semi-permanent mount.

IV. Results Expected: Within 4 days of inoculating the new corn meal agar plate, all the sexual and asexual stages of this fungus can be observed.

V. Cautionary Notes: Basidiobolus ranarum has been described from a number of animal lesions, and has caused human disease. Pregnant women, immunocompromised individuals, and diabetics should excercise caution with this fungus. Even normal individuals should treat the organism as a potential pathogen. In addition, excessive opening of the culture vessels may cause them to be contaminated by normal fungal spore in the air.

VI. References:

Drechsler, C. 1958. Formation of sporangia from conidia and hyphal segments in an Indonesian Basidiobolus. Amer. J. Bot. 45:632-638.

Groff, J.M., A.Mughannam, T.S. McDowell, A. Wong, M.J. Dykstra. 1991. An epizootic of cutaneous zygomycosis in cultured dwarf African clawed frogs (Hymenochirus curtipes) due to Basidiolus ranarum. J. Med. Vet. Mycol. 29:215-223.

FIGURES:

Figure 1: Conidia forming at the tips of conidiophores arising from the mycelium on the agar surface. X ***.

Figure 2: Conidia (a) and Capilloconia (b) stuck to the lid of the Petri dish. X ****

Figure 3: Empty ballistosporic conidia on the Petri dish lid, with emergent germ tube which has given rise to a new ballistosporic conidium. This process can go on for several generations before the process becomes metabolically impossible. X ****

Figure 4: Zygospores on surface of agar from Petri dish culture. Note the short beaks (arrows) representing the gametangia. X****