Dino 9 Workshop Report: Linking Dinoflagellate Life-Cycle Stages Using Single-Cell Isolation And Molecular Typing

This workshop aimed to expose the participants to simple techniques for manipulation of single cells and cysts, with current and emerging DNA technologies to identify the genetic and taxonomic affinities of cyst/cell morphotypes and link different life-cycle stages of dinoflagellates. The workshop was attended by over 45 participants (from 23 countries), ranging from research (Masters and PhD) students through to senior researchers. The workshop kicked off with two short presentations from the workshop leaders. Dr Bolch described and demonstrated the practice of manual mouth pipetting (including a short video) using both stereo- and compound microscopes. Simple techniques to improve success rate of manual manipulations were covered as were ways of minimizing saltwater/media carry over with transfer of single cells to PCR tubes. Dr Percy presented a range of alternative manipulation methods (including video presentations) that avoid mouth pipetting, a practice that is increasingly banned in laboratories where work with human or infectious agents may be carried out in the same areas as lower-risk activities. Following the presentations, all participants practised the demonstrated methods with live dinoflagellate cells using simple mouth pipettes and other devices supplied by the presenters, using a range of equipment made available by the University of Liverpool laboratory and academic staff. Most participants experienced successful manipulation and transfer of single cells during the hour of practice. All participants considered the workshop valuable and that the very simple manipulations could be usefully implemented in their own laboratories for specific research questions and projects. A number of participants also decided to keep their piece of silicon tubing to manufacture a similar device on return to their lab. Toward the end of the workshop, a short presentation from Dr Bolch focused on how some current and emerging technologies (laser-microdissection, flow cytometry, pico-litre droplet manipulation) can be combined with molecular methods (multiple displacement amplification) to achieve both highly selective and automated high-throughput manipulation for molecular and genomic scale analysis of single bacteria through to phytoplankton protistan cells. The workshop presenters thank Dr David Montagnes and lab staff at the University of Liverpool for enthusiastic assistance and efforts to round up suitable equipment to run the workshop. Thanks also go to Dr Joe Taylor (University of Westminster) for his on-the-ground organization and logistics to ensure that the workshop would be a success. The workshop technical notes are reproduced below for a wider audience.