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Multiple ecological and genetic studies of diatom algae require an axenic culture. However, algae-associated bacterial biofilms often form in diatom-produced mucus, both during creation of monoclonal cultures from single cells and during biomass growth, and they may be difficult to remove. In this work, we describe a protocol for removing associated bacteria from a monoclonal culture of Ulnaria danica isolated from Lake Baikal. The axenization strategy involves selecting the latent phase of diatom growth, multiple washes to remove extracellular polymeric substances and bacterial cells, filter deposition, and treatment with antibiotics that are not toxic for diatoms. The absence of bacteria during these stages was controlled by light microscopy with Alcian blue staining for mucus, epifluorescent microscopy with DAPI (4′,6-diamino-2-phenylindole) staining for bacterial DNA, and scanning electron microscopy of the diatom cell surface. High-throughput sequencing of a 16S rRNA fragment, amplified with universal bacterial primers, from total DNA of a final culture failed to detect any bacterial contamination, confirming successful axenization. A detailed comparative description of all stages of our protocol may prove useful in developing axenic cultures of other diatoms for various ecological and genetic studies.

Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom