title: Optimizing the setup of a flow cytometric cell sorter for efficient quantitative sorting of long filamentous cyanobacteria.
abstract: Heterogeneity within natural phytoplankton communities makes it very difficult to analyze parameters at the single-cell level. Flow cytometric sorting is therefore a useful tool in aquatic sciences, as it provides material for post-sort analysis and culturing. Sorting subpopulations from natural communities, however, often requires handling morphologically diverse and complex particles with various abundances. Long particles, such as filament-forming cyanobacteria (>100-μm long), prove very difficult to handle. These potentially toxic organisms are widespread in eutrophic systems and have important ecological consequences. Being able to sort filamentous cyanobacteria efficiently and as viable cells is therefore highly desirable when studying factors associated with their toxicity and occurrence. This unconventional sorting requires extensive user experience and special instrument setup. We have investigated the effect of hydrodynamic and electromechanical components of a flow cytometer, and sorting protocol on the quantitative sorting efficiency of these long particles using two filamentous cyanobacterial strains with average lengths of ∼100 and ∼300 μm. Sorting efficiency ranged from 9.4 to 96.0% and was significantly affected by filament length, sorting envelope, drop delay (dd), and for the long species also by tip size, but not by cycle time. Filaments survived sorting and were not damaged. The optimal settings found for the modular MoFlo® cell-sorter to sort the filaments were a 100-μm flow tip at 30 psi (207 kPa) with a three-droplet envelope in Enrich mode while using an extended analysis time of 17.6 μs and an intermediate plate charge and deflection percentage combination of 3,000 V/60%, combined with a dd 0 for the cultures with 100-μm filaments and dd +1 for the culture with 300-μm filaments. To the best of our knowledge, the filaments up to 1063.5 μm sorted in this study are the longest ever sorted.
authors.affiliation: Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands. m.vandijk@nioo.knaw.nl
authors.forename: Mark A
authors.initials: MA
authors.lastname: van Dijk
authors.forename: Gerald
authors.initials: G
authors.lastname: Gregori
authors.forename: Hans L
authors.initials: HL
authors.lastname: Hoogveld
authors.forename: Machteld
authors.initials: M
authors.lastname: Rijkeboer
authors.forename: Michel
authors.initials: M
authors.lastname: Denis
authors.forename: Anthony
authors.initials: A
authors.lastname: Malkassian
authors.forename: Herman J
authors.initials: HJ
authors.lastname: Gons
mesh_terms: D002448:Cell Adhesion; D002465:Cell Movement; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D000458:Cyanobacteria / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods