Culture method

  1. Medium
  2. Food
  3. Vessel
  4. Subculture method

1. Medium

  1. BLEY medium
  2. LE medium
  3. SE medium
  4. Modified EGG medium
  5. MA medium
  6. M11 medium

1.1. BLEY medium (Barley leaf & Egg-yolk extracted liquid)

BL Solution: 1 pack (3 g) of Barley leaf (Trade name: Oomugi Wakaba Funmatu 100%, Yamamoto Kanpou Seiyaku Co.) is added to 1,000 ml distilled water and boiled for 10 minutes, while stirring. The solution is passed through the qualitative filter paper.

EY Solution: 9 g of crushed yolk of hard-boiled egg is added to 1,000 ml distilled water and boiled for 10 minutes, while stirring. The solution is passed through the qualitative filter paper.

BL and EY Solution are mixed. The mixed solution is dilution with distilled water up to 6,000 ml, and then sterilized by autoclaving.

1.2. LE medium (Lettuce & Egg-yolk extracted liquid)

L Solution: Parts of light green leaves of lettuce is dried at 90 degrees C for about 16-18 hr without scoring. 3 g of the dried leaves is added to 1,000 ml boiling water (9:1 distilled water/tap water) and boiled for 30 minutes, while stirring. The supernatant is passed through sanitary cotton layers.

Selection of lettuce

Dried lettuce

Boiling

E Solution: 3 g of crushed yolk of hard-boiled egg is added to 1,000 ml water (9:1 distilled water/tap water) and boiled for 30 minutes, while stirring. The supernatant is passed through cotton layers.

Equal quantities of L and E Solution are mixed. pH of the solution is adjusted to 6.8 with 1N NaOH or 1N HCl. Next the solution is sterilized by autoclaving.

Notes

Lower the pH while keeping refrigerated.

Chemical analysis (n=1)

TOC: approx. 370 mg/L, T-N: 31 mg/L, NH4-N: 4.7 mg/L, NOx-N: 9.7 mg/L, T-P: 6.5 mg/L, PO4-P: 5.5 mg/L.

1.3. SE medium (Sludge extracted liquid)

We use activated sludge of municipal treatment plant indicating the degree of treatment efficiency. MLSS of the sludge is adjusted about 5,000 mg/L, then autoclaved (121 degrees C, 30 minutes). The supernatant is passed through cotton batting. Next, pH of the solution is adjusted to 6.8-7.0 with 1N NaOH or HCl. The solution is sterilized by autoclaving.

1.4. Modified EGG medium

A Solution: Glucose 0.15 g, Na-acetate 0.15 g, (NH4)2SO4 0.05 g, MgSO4/7H2O 0.025 g, KCl 0.025 g, CaCO3 0.025 g, Agar-extract 100 mL (20 g of agar powders is added to 1,000 mL distilled water, and then keep still overnight in room temperature. The supernatant is filtered by GFC.), distilled water 900 mL.

B Solution: 0.2 M Phosphate buffer 5 ml/L. A-liquid 61 mL and B-liquid 39 mL mixed and then autoclaved.

  • A-liquid: Na2HPO4/12H2O 71.64 g/L
  • B-liquid: NaH2PO4/2H2O 31.21 g/L

    C Solution: FeCl3/6H2O 0.0005 g/L.

    D solution: Vitamin B12 5x10-6 g/L, Vitamin B1 1x10-4 g/L (Keep the stock solution keep in a deep freezer. Before use, sterilize by filtration through a milipore filter (pore diameter: 0.22 um).

    Filtration tools

    E Solution: Stigmasterol 0.01 g/L. Stigmasterol 50 mg is soluble 10 mL ethanol (99.99%).

    A Solution is sterilized by autoclaving, then B, C, D, E solution are added.

    1.5. MA medium

    Stock solution: Ca(NO3)2/4H2O 5 mg, KNO3 10 mg, NaNO3 5 mg, Na2SO4 4 mg, MgCl2/6H2O 5 mg, Beta-Na2glycerophosphate 10 mg, Na2EDTA 0.5 mg, FeCl3/6H2O 0.05 mg, MnCl2/4H2O 0.5 mg, ZnCl2 0.05 mg, CoCl2/6H2O 0.5 mg, Na2MoO4/2H2O 0.08 mg, H3BO4 2 mg, Bibine 50 mg, distilled water 100 mL. Final pH should be 8.6.

    1.6. M11 medium

    Stock solution: NaNO3 10 mg, K2HPO4 1 mg, MgSO4/7H2O 7.5 mg, CaCl2/2H2O 4 mg, Na2CO3 3 mg, FeSO4/7H2O 0.1 mg, Na2EDTA 2H2O 0.1 mg, distilled water 100 mL. Final pH should be 8.0.

    2. Food

    2.1. Bacteria

    2.2. Algae (for algal-feeders)

    Anabena No.1, No.77 (NIES)
    Stock culture: MA-medium or M11-medium. 25 degrees C, shaking, 3 weeks, about 2,000 lux.

    2.3. Protozoa (for carnivorous protozoa)

    Tetrahymena pyriformis (Tetra-1)

    3. Vessel


    4. Subculture method

    Incubation temperature 10-30 degrees C.

    Flowchart