Directions for pure culture

Cultures of Batrachochytrium will be sent on nutrient agar in sealed Petri plates.  Upon receipt, transfer small chunks of culture to several containers of 1% T broth to serve as stock cultures. If you intend to use the culture repeatedly it is a good idea to cryopreserve some.  See directions in (Boyle, D. G. et al. (2003). Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes. Diseases Of Aquatic Organisms 56: 59–64).

Stock cultures of Batrachochytrium remain viable longer when grown in broth.  Place 75 mL of broth into 150 mL screw-capped flasks–there’s nothing sacred about size–use larger or smaller containers depending on what is available.  Because of the danger of contamination, I use screw-capped vessels and keep duplicates.  Incubate at 23 C or below.  For long-term storage (at least 3 months), place at 4-5 C once growth is evident. When growing to produce zoospores for inoculum or other purposes, grow on 1% tryptone agar medium.

Production of zoospores: Grow Batrachochytrium in broth until clumps of thalli are visible to the eye.  Use a sterilized Pasteur pipette to add 1/2 to 3/4 mL of this broth culture to tryptone agar in 9 cm culture dishes.  Leave inoculated dishes open in laminar flow hood until the added broth is dry; replace covers on dishes and either seal plates with Parafilm® or place in plastic sleeve.  Incubate in an incubator set between 15 and 23 C; the potential harvest period is longer if plates are kept in plastic sleeves at 15 C.  After 3–10 days, look for active zoospores around the periphery of the fungal colonies by inverting the dishes on the stage of a compound microscope and examining with the 10 X objective.

Harvest zoospores: Flood plates with 2–3 mL of sterile distilled water and decant after about 30 minutes to collect zoospores. Zoospore concentration can be measured by optical density or by counting killed spores on a hemocytometer. I kill zoospores by adding an equal volume of Lugol’s iodine solution. Zoospores may stay motile (thus infective) for up to 24 hours, however, most encyst before 24 hours.

Biosafety: This organism can be lethal to amphibians. Although thorough drying will probably kill it, leave nothing to chance.   Autoclave all materials that contain or have come into contact with the fungus before disposal.  If Batrachochytrium is used to inoculate amphibians, kill and incinerate or fix all exposed animals after experiments.  Be sure that cages, water, and other material in cages are disinfected at the end of experiments. Do not dump potentially contaminated material in the trash or down the drain without first treating it (heat or bleach) to kill the fungus.

Sterilize pipettes

Place cotton plug in wide ends of pipettes, place pipettes in a pipette sterilization container or wrap in heavy aluminum foil, and autoclave for 30 minutes.

Recipes

Tryptone agar
10 g tryptone
10 g agar
1000 mL distilled water

Tryptone broth
10 g tryptone
1000 mL of distilled water