In vitro culture of algae

Isolation of Symbiotic Chlorella

1. Green hydra were starved for 1 week in antibiotic-containing Hydra medium⁽¹⁾.
2. Approximately 50 antibiotic-treated polyps were transferred into a 1.5-ml microcentrifuge tube and washed three times with antibiotic-containing Hydra medium.
3. Remove the Hydra medium, add 800 μl of 0.25% SDS in distilled water (0.5% Tween-20 can be used as an alternative), and immediately homogenize the polyps by pipetting with a P1000 micropipette to disrupt the Hydra tissues. Homogenization should be completed as quickly as possible because prolonged exposure reduces Chlorella viability.
4. Centrifuge at 3,000 × g for 1 min at room temperature.
5. Wash the pellet with 0.05% SDS in distilled water (0.1% Tween-20 can be used as an alternative).
6. Centrifuge at 500 × g for 5 min at room temperature.
7. Discard the supernatant and wash the pellet with antibiotic-containing Hydra medium.
8. Centrifuge at 500 × g for 5 min at room temperature.
9. Discard the supernatant. The pellet was used for subsequent culture as the isolated symbiotic Chlorella.
10. The isolated Chlorella cells were washed with sterile modified Bold's Basal Medium⁽²⁾ supplemented with 0.5% glucose, 1.2 mg/L vitamin B1 (thiamine hydrochloride), and 0.01 mg/L vitamin B12 (cyanocobalamin), and incubated for two days in modified Bold Basal Medium with 50 mg/L ampicillin and streptomycin.
11. The isolated symbiotic Chlorella cells were cultured according to the protocol described in the Materials and Methods section.

(1) Antibiotic-containing Hydra Medium

Hydra medium or Volvic water supplemented with 50 mg/L each of ampicillin, rifampicin, streptomycin, and neomycin.

(2) Formulation of Modified Bold's Basal Medium

Stock solutions

Preparation of Modified Bold's Basal Medium

To prepare 1 L of Modified Bold's Basal Medium, add 10 ml each of stock solutions 1–6 and 1 ml each of stock solutions 7–10 to distilled water, adjust the final volume to 1 L, and sterilize the medium by autoclaving.