Culture method

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

Medium
  1. BLEY medium
  2. LE medium
  3. SE medium
  4. Modified EGG medium
  5. MA medium
  6. M11 medium
[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 solutions are mixed. The mixed solution is dilution with distilled water up to 6,000 ml, and then sterilized by autoclaving.

[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. 3g 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 cotton batting.
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 batting.
Equal quantities of L and E solutions 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 (one time) 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.

[SE medium] (Sludge extracted liquid)
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.

[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: Na2HPO4/12H2O 71.64 g/L
B-liquid: NaH2PO4/2H2O 31.21 g/L
A-liquid 61mL and B-liquid 39 mL mixed and then autoclaved.
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)
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.

[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.

[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.


Food

[Bacteria]
For bacteria-feeders: These bacteria are mixed for food.
Micrococcus luteus (IAM 1313)
Acinetobacter calcoaceticus (IAM 1517)
Flavobacterium lutescens (IAM 1667)
Pseudomonas ovalis (IAM 1002)
Bacillus cereus (IAM 1029)
Klebsiella pneumonaie (IAM 1102)
Escherichia coli (IAM 1239)
Bacillus sabtilis (IAM 1069)

Stock culture: Standard agar medium, slant, 10 degrees C.
Liquid culture: LE medium, Tube, 20-30 degrees C.

For filamentous bacteria-feeders:
Sphaerotilus natans (SY-1) (Kohno strain)
Stock culture: Slant, 20 degrees C, 1/2CG medium (Bact-Casiton 0.25 g/L, Glucose 0.5 g/L, Yeast extract 0.25 g/L, tap water keeping one overnight in room temperature 1 L)
Mass culture: 30 degrees C, 300 mL flask (liquid volume 100 mL), culture period 1 day, centrifugation condition (10,000 rpm, 5 minutes), washing solution (1/750 M phosphate buffer).

Type 021N (T2-1, SNR-3) (Kohno strain)
Stock culture: Slant, 25 degrees C, EGG medium (Glucose 0.3 g/L, Na-acetate 0.3 g/L, (NH4)2SO4 0.1 g/L, MgSO4/7H2O 0.05 g/L, KCl 0.05 g/L, CaCO3 0.05 g/L, Agar 10 g/L, distilled water 1 L, 0.2M phosphate buffer solution 5 mL/L, FeCl3/6H2O 5x10E-4 g/L, Vitamin B12 5x10E-6 g/L, HCl-Thiamine (B1) 1x10E-4 g/L).
Mass culture: EGG-medium (Glucose 0.3 g/L, Na-acetate 0.3 g/L, (NH4)2SO4 0.1 g/L, MgSO4/7H2O 0.05 g/L, KCl 0.05 g/L, CaCO3 0.05 g/L, 0.2M phosphate buffer solution 5 ml/L, FeCl3/6H2O 5x10E-4 g/L, Vitamin B12 5x10E-6 g/L, HCl-Thiamine (B1) 1x10E-4 g/L) + Agar layer (0.5 g/50 mL), 25 degrees C, static, 7-10 days.
Pipet-washing: Washing solution (1/750 M phosphate buffer).
Ultrasonics, 10 degrees C stock.
Reference: T. Kohno (1988), Morphology, physiology, and nutrition of a sulphur-oxidizing filamentous organism isolated from activated sludge, Wat. Sci. Tech.,20, 241-247.

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

For carnivorous protozoa:
Tetrahymena pyriformis (Tetra-1)
Flagellata


Vessel
Tube , Petri dish , 300ml elenmyer flask
Subculture method
Incubation at 10-30 degrees C.