AXENIC CULTURES OF ACETABULARIA (CHLOROPHYTA): A DECONTAMINATION PROTOCOL WITH POTENTIAL APPLICATION TO OTHER ALGAE1

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Abstract
We developed an easy, reliable decontamination protocol for caps of Acetabularia acetabulum (L.) Silva; with minimal labor hundreds of caps can be decontaminated. In addition, cysts isolated from these caps do not exhibit dormancy and can be used immediately to establish large populations of axenic cell cultures. This method consists of three successive incubations: 1) proteinase K/sodium dodecyl sulfate/1,3‐bis[tris(hydroxymethyl)‐methylamino]propane for 1 h, 2) mild silver protein for 5 min, and 3) an antibiotic solution (neomycin, chloramphenicol, nystatin, ampicillin, streptomycin)for 3 days. This protocol eliminates bacterial, algal, fungal, and yeast contaminants. It is useful for decontaminating caps from lab cultures and caps collected from the wild and may also be effective in decontaminating the reproductive structures of other algae. Cyst dormancy was reduced from 15 weeks to less than 1 week, which represents a 40% reduction of the life cycle of A. acetabulum. We routinely obtained 90‐100% gamete release from cysts 3‐14 days after they were made axenic. The component of our culturing methods that allows gamete release without a “dormant” period is unknown.