Cyanobacteria filaments up to 1063.5 μm sorted with a Moflo cell sorter

mardi 15 juin 2010

Van dijk, M.,Grégori, G.,Hoogveld, H.,Rijkeboer, M.,Denis, M.,Malkassian, M.,Gons, H. (2010) Optimizing the setup of a flow cytometric cell sorter for efficient quantitative sorting of long filamentous cyanobacteria. Cytometry 77A, Issue 10:905–995

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 and for the long species also to 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 3 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 3000 V / 60 %, combined with a drop delay 0 for the cultures with 100 μm filaments and drop delay +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.

Key terms : Flow cytometry, quantitative, sorting, filaments, cyanobacteria