Sporulation and sterilization method for axenic culture of Gelidium canariensis

A sporulation and sterilization procedure was used to establish axenic cult ures of sporelings of Gelidium canariensis. Sporangial branchlets excised f rom the thallus were rinsed in distilled water twice and in 1% sodium hypoc hlorite (2 min). The branchlets were cultivated overnight in multiwell plat es with 0.3 mi of autoclaved seawater to promote spore liberation in 90% of the cultivated branchlets. The branchlets were transferred to an antibioti c solution made of ampicillin, penicillin, rifampicin, nystatin (0.2 mg ml( -1) each) and 0.1 g ml(-1) of GeO2 in liquid PES for 45 days, during which clusters of spores (85-100 spores) were observed on the surface of the bran chlet. After 55 days, they became axenic sporelings with the prostrate and erect system characteristic of Gelidium canariensis. (C) 1999 Elsevier Scie nce B.V. All rights reserved.