Axenic clonal cultures of filamentous brown algae: Initiation and maintenance

The increasing number of molecular and biotechnological studies involving brown algae has led to a growing demand for bacteria-free cultures. We describe here simple methods to establish axenic clonal cultures of filamentous marine brown algae. Thallus fragments are exposed on agar plates to commercially available paper disks loaded with defined concentrations of antibiotics. Regenerating fragments are isolated and used to initiate axenic algal cultures in liquid medium or on agar. We found the following agents to be tolerated by algae and active against marine bacteria: ciprofloxacin, chloramphenicol, rifampicin, polymyxin B, and kanamycin. Axenic agar cultures are maintained by serial transfer on agar, or re-introduced into liquid medium. Although we recommend transfer of agar cultures in 2-3 month intervals, we have observed survival times on agar of up to 2 years without attention.