Solid Growth Substrate for Symbiodiniaceae - IBEMicrobeHub/culturing GitHub Wiki

Protocol for Developing a Solid Growth Substrate for Symbiodinium Cultures

Objective

To isolate individual clones and establish new "axenic" cultures of Symbiodinium using a solid growth substrate supplemented with an effective antibiotic cocktail.

Materials

  • Agar
  • f/2 medium
  • Kanamycin
  • Ampicillin
  • Streptomycin

Method

Preparation of Solid Growth Substrate

  1. Prepare Agar Plates:
    • Mix 1% f/2 medium with agar to form the base of the growth substrate.
    • Sterilize the mixture by autoclaving and allow it to cool to approximately 50°C before proceeding to the next step.

Antibiotic Supplementation

  1. Add Antibiotics:
    • Supplement the agar with the following antibiotics to prevent contamination and support the growth of Symbiodinium:
      • Kanamycin: 50 µg/mL
      • Ampicillin: 100 µg/mL
      • Streptomycin: 50 µg/mL
    • Ensure that antibiotics are thoroughly mixed into the agar to distribute evenly.

Plate Pouring and Culture Establishment

  1. Pour Plates:

    • Pour the antibiotic-supplemented agar into sterile petri dishes under aseptic conditions to avoid any contamination.
    • Allow the plates to solidify at room temperature.

  2. Inoculation:

    • Inoculate the solidified agar plates with Symbiodinium cultures using a sterile loop or pipette tip.
    • Spread the culture gently to ensure individual colonies can develop.

  3. Incubation:

    • Incubate the plates in conditions suitable for Symbiodinium growth until colonies are visibly established and free of any signs of contamination.

Observation

  • Monitor the plates for signs of contamination and the health of Symbiodinium cultures.
  • Record the effectiveness of the antibiotic cocktail in promoting the growth of axenic cultures.

Notes

  • The chosen concentration of antibiotics has been found most effective at eliminating visual signs of contamination without harming various Symbiodinium strains in culture.
  • Adjustments to antibiotic types and concentrations may be necessary based on specific strain sensitivity and experimental conditions.