Improved Methods for Bulk Cultivation and Fixation of Loxodes Ciliates for Fluorescence Microscopy

Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physi-ological 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 fix-atives 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 formalde-hyde 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.(c) 2022 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Automated summary

In this study, methods for growing and harvesting Loxodes cells in bulk with liquid soil extract medium were described, along with a new fixative called ZFAE that effectively fixes Loxodes cells. This allows for long-term maintenance, large-scale growth, and modern cell biology investigations of Loxodes monoclonal strains in laboratory conditions.