{"title":"Improved Methods for Bulk Cultivation and Fixation of Loxodes Ciliates for Fluorescence Microscopy","authors":"Brandon Kwee Boon Seah, Christiane Emmerich, Aditi Singh, Estienne Carl Swart","doi":"10.1016/j.protis.2022.125905","DOIUrl":null,"url":null,"abstract":"<div><p><em>Loxodes</em> is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we describe methods to grow and harvest <em>Loxodes</em> cells in bulk with liquid soil extract medium, as well as a new fixative called ZFAE (zinc sulfate, formaldehyde, acetic acid, ethanol) that can fix <em>Loxodes</em> cells more effectively than buffered formaldehyde or methanol. We show that ZFAE is compatible with immunofluorescence and the nuclear stain DAPI. <em>Loxodes</em> is thus now amenable to long-term maintenance, large-scale growth, and modern cell biology investigations of monoclonal strains in laboratory conditions.</p></div>","PeriodicalId":20781,"journal":{"name":"Protist","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2022-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1434461022000505/pdfft?md5=3a0e3858784e9d921f16f284c467c601&pid=1-s2.0-S1434461022000505-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protist","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1434461022000505","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we describe methods to grow and harvest Loxodes cells in bulk with liquid soil extract medium, as well as a new fixative called ZFAE (zinc sulfate, formaldehyde, acetic acid, ethanol) that can fix Loxodes cells more effectively than buffered formaldehyde or methanol. We show that ZFAE is compatible with immunofluorescence and the nuclear stain DAPI. Loxodes is thus now amenable to long-term maintenance, large-scale growth, and modern cell biology investigations of monoclonal strains in laboratory conditions.
期刊介绍：
Protist is the international forum for reporting substantial and novel findings in any area of research on protists. The criteria for acceptance of manuscripts are scientific excellence, significance, and interest for a broad readership. Suitable subject areas include: molecular, cell and developmental biology, biochemistry, systematics and phylogeny, and ecology of protists. Both autotrophic and heterotrophic protists as well as parasites are covered. The journal publishes original papers, short historical perspectives and includes a news and views section.
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