Protocol

Isolation of Single Choanoflagellate Cells from Field Samples and Establishment of Clonal Cultures

¹Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94704, USA
²Department of Integrative Biology, University of California, Berkeley, Berkeley, CA 94704, USA
³Canadian Institute for Advanced Research, Toronto, Ontario M5G 1Z8, Canada
⁴Department of Biology, University of York, York YO10 5YW, United Kingdom
⁵School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

⁶Corresponding author (b.s.c.leadbeater{at}bham.ac.uk)

This article is also available in Emerging Model Organisms: A Laboratory Manual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.

INTRODUCTION

Choanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes how to enrich freshly collected field samples and isolate single choanoflagellate cells. Two methods are commonly used: isolation by micropipetting single cells and isolation by dilution. The two methods are complementary and each has its advantages and disadvantages. The micropipette technique requires considerable experience. However, provided a single cell has been isolated, any culture arising from it will be clonal. The dilution technique does not require the same level of skill, but single-cell isolation is not ensured. For the latter technique, repeating the isolation procedure using higher dilutions is advised to ensure that only one cell is the basis of an established culture.