I. Objectives: To observe the production of asexual zoospores, cysts and vegetative hyphae by Saprolegnia.

II. Materials:

• Saprolegnia diclina (ATCC 52719)
• sterile 250 ml flasks
• 100 rpm shaker platform or water bath
• Griffin's (1978) GY broth:
  5 g glucose
  2.5 g yeast extract (dissolved in 1 L distilled water and autoclaved)
  
• Griffin's (1978) SM medium:
  0.25 mM CaCl2
  0.25 mM KCI (made up with distilled water then autoclaved)
  
• Petri dishes containing CM+ agar:
  17 g Difco corn meal agar
  2 g glucose
  1 g yeast extract (dissolved in 1 L distilled water and autoclaved)
  
• sterile 10 or 25 ml transfer pipets and bulbs
• compound light microscope
• plastic Petri dishes
• microscope slides
• cover slips

III. Procedure:

• Inoculate plate of CM+ with Saprolegnia. Grow for 1 week at room temperature.
• Cut 2-3 mm plugs of agar from the growing front of the mycelium and place in 30 ml of GY broth in a 250 ml flask on a 100-120 rpm shaker at room temperature for three days.
• After 3 days, remove agar plugs that will be covered with fuzzy mycelial growth and rinse several times in SM medium. Next, transfer these mycelial wefts to 20 ml of fresh SM in 100 mm plastic Petri dishes and leave at room temperature without shaking.
• Between 16 and 24 hours later, zoospores will begin to be released from the zoosporangia.

IV. Results: Within 16-24 hours after the mycelium was transferred from GY to SM medium, zoospores should be readily evident emerging from zoosporangia (the darkened areas of hyphal tips). In addition, encysted zoospores will be scattered across the bottom of the Petri dish and attached to hyphae. After 24-48 hours, cysts will germinate by producing delicate hyphae.

V. Cautionary Notes: Even though no human diseases have been associated with Saprolegnia sp., the organism should be treated as a pathogen (observe proper sterile techniques during handling and disposal of the cultured material). This procedure will work equally well with Achlya or Aphanomyces sp.

VI. References: