Testing for axenicity

At ANACC we use 3 methods for testing axenicity in strains. Bactopeptone broth and SWAV plates are prepared using the recipes below. We also use commercial Petrifilm Aerobic Count plates produced by 3M.

 

Bactopeptone Broth 0.1%

• Bacteriological peptone 1g
• 0.22µm filtered seawater or Culture growth medium 1L

Mix well and dispense ~10mL of Bactopeptone Broth into MacCartney bottles. Sterilise by autoclaving at 121°C for 20mins.

To test for absence of bacteria – aseptically add ~0.1mL of microalgal culture to sterile Bactopeptone Broth.

Incubate at 25°C for 5-7 days.

The broth should be clear if no bacteria present

 

SWAV (seawater agar with f2 vitamins)

• Yeast extract 1.0g
• Bacteriological peptone 1.0g
• Bacteriological agar 15.0g
• f2 vitamin stock 2.5mL
• 0.22µm filtered seawater 1L

Add yeast extract, peptone and agar to the filtered seawater and aseptically add sterile f2 vitamin stock solution.

Sterilise by autoclaving at 121°C for 20mins. Pour agar into petri dishes (90mm) and allow to cool and solidify.

Aseptically add ~0.1mL of culture to SWAV plate and spread. Seal petri dish with parafilm and incubate for at least 5 days at 25°C.

The plate should be free of bacterial or fungal colonies for axenic cultures.