Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom
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 w...
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MDPI AG
2020-10-01
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| Online Access: | https://www.mdpi.com/1424-2818/12/10/382 |
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| author | Yulia Zakharova Artem Marchenkov Nadezhda Volokitina Aleksey Morozov Yelena Likhoshway Mikhail Grachev |
| author_facet | Yulia Zakharova Artem Marchenkov Nadezhda Volokitina Aleksey Morozov Yelena Likhoshway Mikhail Grachev |
| author_sort | Yulia Zakharova |
| collection | DOAJ |
| description | 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 <i>Ulnaria danica</i> 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. |
| first_indexed | 2024-03-10T15:51:54Z |
| format | Article |
| id | doaj.art-93ceb8d7bcb0460a8361852be9bd0e1f |
| institution | Directory Open Access Journal |
| issn | 1424-2818 |
| language | English |
| last_indexed | 2024-03-10T15:51:54Z |
| publishDate | 2020-10-01 |
| publisher | MDPI AG |
| record_format | Article |
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| spelling | doaj.art-93ceb8d7bcb0460a8361852be9bd0e1f2023-11-20T15:59:56ZengMDPI AGDiversity1424-28182020-10-01121038210.3390/d12100382Strategy for the Removal of Satellite Bacteria from the Cultivated DiatomYulia Zakharova0Artem Marchenkov1Nadezhda Volokitina2Aleksey Morozov3Yelena Likhoshway4Mikhail Grachev5Department of Cell Ultrastructure, Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, RussiaDepartment of Cell Ultrastructure, Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, RussiaDepartment of Cell Ultrastructure, Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, RussiaDepartment of Cell Ultrastructure, Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, RussiaDepartment of Cell Ultrastructure, Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, RussiaDepartment of Cell Ultrastructure, Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, RussiaMultiple 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 <i>Ulnaria danica</i> 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.https://www.mdpi.com/1424-2818/12/10/382xenic cultivating diatomalgal–bacterial associationextracellular polymeric substationAlcian blue staining16S rRNA gene sequencingaxenic culture |
| spellingShingle | Yulia Zakharova Artem Marchenkov Nadezhda Volokitina Aleksey Morozov Yelena Likhoshway Mikhail Grachev Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom Diversity xenic cultivating diatom algal–bacterial association extracellular polymeric substation Alcian blue staining 16S rRNA gene sequencing axenic culture |
| title | Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom |
| title_full | Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom |
| title_fullStr | Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom |
| title_full_unstemmed | Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom |
| title_short | Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom |
| title_sort | strategy for the removal of satellite bacteria from the cultivated diatom |
| topic | xenic cultivating diatom algal–bacterial association extracellular polymeric substation Alcian blue staining 16S rRNA gene sequencing axenic culture |
| url | https://www.mdpi.com/1424-2818/12/10/382 |
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