Incubatuin of phytoplankton

Incubation of phytoplankton (Nannochloropsis sp., Chaetoceros sp. and T-ISO)

Masatoshi Futagawa/CORDUNAP

January 17, 2013

Phytoplankton are primary live feeds for shellfish larva and rotifer. And rotifer as live feeds for finfish larva that contains HUFAs which is essential to marine fish larva. Thus, constant phytoplankton production without contamination of protozoans is one of key factors to produce the larvae. Recently, there are some substitute products of phytoplankton such as dry phytoplankton and enrichment material which HUFAs concentrated. Even so, live phytoplankton is the best material for larvae culture as of present. I will introduce typical phytoplankton incubation method that is practiced for scallop larva culture in our hatchery.

1.    Medium preparation

1.1.  Seawater is filtered by fiber filters (500, 100, 50, 10, 1 and 0.5 µm) and sterilized by UV at hatchery. The water is filtered again by membrane filter (0.45 µm, Advantech) with vacuum pump in a laboratory. The membrane is use for filtering 1 L water only.

  

1.2.  Filtered water (2 L) mixed with f/2-Si medium (see list below) by pipet to prepare culture medium.

NaNO3 (75.0 g/L dH2O)	1.0 ml
NaH2PO4·H2O (5.0 g/L dH2O)	1.0 ml
f/2 Trace Metal Solution	1.0 ml
f/2 Vitamin Solution	0.5 ml
Filtered seawater	1.0 L

1.3.    Cultured medium (2 L) is poured in small bottles (200 ml) after the bottles are washed by the medium. The bottles are closed by cotton cap and aluminum foil. Those culture medium are disinfected by autoclave (121 ºC, 15 min.). It is possible to use pressure cooker as substitute of autoclave for disinfection.

 

2.    Inoculation 1^st (200 ml)

When inoculation, hands are cleaned by alcohol (60 %) with latex gloves to avoid contamination before inoculation. Plankton stocks (120 ml) are inoculated to the incubation bottles (200 ml) inside clean bench. Those bottles are kept without strong light 4 days by shaking gently more than twice a day. Incubation room was equipped by air conditioner to keep constant temperature. After medium color turn to plankton color, giving strong lights and culture 2 weeks with shaking gently. After 2 weeks from inoculation, the plankton time to extend to 1.5 L volume.

3.    Inoculation 2^nd (2 L)

Bloomed plankton (90 ml) is inoculated to medium (1.5 L) at culture bottle (2 L) and gives strong aeration and light to increase propagation at incubation room. In addition to that, a part of the phytoplankton (30 ml) is reused for new plankton stock in order to refresh stock. It is important not to transfer sediment at bottle bottom, dead plankton, which makes bacterial contamination. Within 8 to 9 days, the plankton has time to extend to 10 L volume.

4.    Incubation 3^rd (10 L)

Filtered sea water, pass same sets of filters as medium preparation, filled 10 L in culture bottle (20 L) with aeration and add commercial chlorine (1 ml/L) to disinfect water. After 2 to 24 hours, the water neutralized by sodium thiosulfate pentahydrate (0.16 g/L) and keep 30 minutes. Add chemicals of f/2-Si medium to prepare culture medium and give strong aeration. The plankton (1.5 L) is inoculated to the medium at culture bottle without sediment. Optionally, two of 2^nd inoculated plankton (180 ml) is inoculated to 15 L of culture water. From this incubation, operate at large incubation room which equipped by air conditioner to keep particular temperature and give strong light. The plankton blooms after 6 to 7 days.

5.    Inoculation 4th (15 L)

Culture mediums are prepared as Incubation 3rd, give strong aeration and light. Bloomed plankton (10 L) is inoculated to two of culture bottles (15 L x 2) without sediment. The plankton grow to high density after 6 to 7 days and ready to feed. It is important to limit operators who care plankton and minimize contamination.