Comparative Physiology @ Guelph
Unicellular algae culturing protocol
1. Millipore filter (0.45 mm) seawater (no more than 2-3 days before sterilization).
2. Clean Corning culture bottles using ‘E’ brush and water. If algal film present, rinse with small amount of bleach in tap water. Rinse well. If algal film persists, wash with 2-4 ml of 0.3-0.5% potassium dichromate in concentrated sulfuric acid. Rinse well.
Culture medium preparation
3. Sterilize bottles and caps by immersion in bleach and tap water (10 drops bleach in 2 gal in plastic tub). Rinse with lots of running tap water (5-10 times). Dry outside of bottles completely.
4. Fill large (1 liter) bottles to 900ml with MPFSW, fill small (250 ml) bottles to 200ml with MPFSW.
5. Add Fritz’s f/2 nutrient medium concentrate.
5A. Add 25 ml of A and 25 ml B nutrient concentrate to each small bottle (200ml medium).
5B. Add 120 ml of A and 120 ml B nutrient concentrate to each large bottle (900ml medium).
6. Sterilize loosely capped in microwave oven. 3-5 min per large bottle; 1-1.5 min per small bottle. Observe bottles; DO NOT BOIL.
Ref: Keller, M.D., et al. 1988. JEMBE 117:279-283.
7. Cap tightly and cool to room temperature. Sterilized medium can be stored in the dark for 2 weeks before inoculation.
8. Inoculate with actively growing culture (see below). Cap with aeration and place near culture lights.
Small cultures (200 ml)
– use only to maintain clean stocks.
– start new culture twice weekly using 1-5 ml inoculum from healthy stock culture.
Large cultures (900 ml)
– use only as working stocks for larval feeding.
– start as many new cultures as are needed, 3-5 days before anticipated use, using 100 ml inoculum from older stock culture.
Light: 24h light