An Improved Technique for Sorting Developmental Stages and Assessing Egg Viability of Globodera pallida using High-Throughput Complex Object Parametric Analyzer and Sorter.

Abstract

The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer designed for analyzing, sorting, and dispensing objects of varying sizes. We explored the potential of using this instrument to analyze and sort various developmental stages and egg viability of Globodera pallida. Cysts were successfully examined and sorted from debris by optimizing side-scatter and red-fluorescence parameters on the COPAS. We were able to separate eggs and second-stage juveniles from samples of mixed population using extinction and time of flight. Separation of live and dead eggs was examined following staining eggs with SYTOX Green and application of time of flight and green peak height. Data were compared with a commonly used viability assay by which eggs were stained with Meldola's Blue and examined by a microscope. COPAS proved to be effective in assessing viability by detecting two separate gates: live eggs having green fluorescence peaks <190 and dead eggs with the peaks >190. The application of COPAS in combination with SYTOX Green detected a greater number of live eggs than the Meldola's assay, suggesting that SYTOX Green provided an overestimate of live eggs. COPAS noticeably increased the accuracy and reduced the time required for screening and analyzing nematode populations.