Sources

• isolation of single species from a plankton tow
  
  
• ordering from various sources:
  
  • CCMP
    
  • UTEX
    
  • Other Algae Collections
    
  • Univ of Utah (CISE)
    
  • Sunnyside Seafarms (for more info on this source see "Growing dinoflagellates at home")
    

Basic Requirements

The following is a basic protocol for growing autotrophic, tropical to temperate marine dinoflagellates. We grow our cultures in Percival incubators on 12 hour light-dark cycles set at 18 °C with light levels at 40-140 µmoles/m²-sec provided by several cool white fluorescent bulbs. The cultures are kept in various-sized, sterile Erlenmeyer flasks, ranging from 250 ml to 2.8 l, using cotton gauze as stoppers. We make F/2 media following the recipe from Guillard, R.R.L. and J.H. Ryther (1962), Can. J. Microbiol. 8:229-239. F/2 refers to enriched seawater that has been shown to be the ideal growth medium for phytoplankton. Our cultures are transferred in a sterile environment into new (sterile) F/2 media every 3 weeks (about 1/4 volume of an old culture is used to inoculate a new culture). Cultures are swirled everyday (except on day of transfer) to prevent clumping of cells and to keep nutrients well mixed.

F/2 Recipe

To 4 l of 0.22 µm filtered open ocean seawater (or artificial seawater) add:

2 ml 15% NaNO₃

2 ml 1% NaH₂PO₄

2 ml trace metals stock

2 ml vitamin stock

10 ml soil extract (re-autoclave after each use)

Fill the various sized flasks each to about 1/2 capacity, then plug them with gauze stoppers and autoclave (steam sterilize) for 20 minutes. Remove F/2 promptly from autoclave (any longer than 20 minutes will cause precipitation). Let sit for 24 hours in incubator to cool.

Tips if you have problems with precipitation (some precipitation is normal)

(1) Autoclave for only 20 minutes, with no dry cycle.
(2) Autoclave just the water, then add supplements.
(3) Silicon is not required for dinoflagellate cultures.
(4) Try filter sterilization instead?

VITAMINS	Concentration	to 500 ml of dist. H2O add:
Thiamine	1 mg/ml	0.5 g
Biotin	1 µg/ml	0.5 mg
Vitamin B12	1 µg/ml	0.5 mg
2. Trace Metals Stock Solution
METALS	Concentration	to 100 ml of dist. H2O add:

FeEDTA	2.34 mM		1000.0 mg
CuSO4•5H2O	8 µM		1.86 mg
ZnSO4•7H2O	15.4 mM		4.4 mg
CoCl2•6H2O	8.6 µM		2.0 mg
MnCl2•4H2O	184 µM		36.0 mg
Na2MoO4•2H2O	5.2 µM		1.26 mg

3. 15% NaNO₃: 15 g NaNO₃ in 100 ml nanopure dist. H₂O

4. 1% NaH₂PO₄: 1 g NaH₂PO₄ in 100 ml nanopure dist. H₂O

5. Soil Extract:

Collect dirt from oaky area or use "rich looking" top soil. Sieve 3-4 handfuls of dirt through a screen. Add dirt to 1 l nanopure dist. H₂O in a large Erlenmeyer flask. Autoclave 20 minutes. Let sit overnight. Pour off supernatant and centrifuge 10 minutes. Pour supernatant through cheesecloth (Repeat centrifugation and filtering if needed). Divide into subsamples (30-40 ml) and store frozen.

Washing Dino Culturing Dishes

• Day 1: After discarding old cultures, wash the flasks with Liquinox detergent, hot tap water, and a scrub brush. Scrub inside of flask well with brush. Rinse excess soap out with tap water.
  
  
• Put one drop of Liquinox in flask and fill to brim with tap water, being careful not to have bubbles around mouth because they will crust. Let flasks soak overnight.
  Day 2: Empty flasks and fill with enough 10% HCl solution to cover typical culture depth in that flask. These flasks should be kept under the fume hood. Let these flasks sit overnight.
  
  
• Day 3: Remove the flasks from the acid bath and scrub out any residue using a designated "acid only" brush. Rinse the flasks 10 times with tap water, then 3 times with DI water. Let air dry. Store in a dry place until flasks are used.

Our Cultures
James Case's lab at UCSB is currently growing:

• Gonyaulax polyedra (aka Lingulodinium polyedrum)
  
• Pyrocystis fusiformis  (picture)
  
• Pyrocystis lunula
  
• Glenodinium sp. (not a bioluminescent species)