Preparation of Cuvette‐Based Sorters for Sorting Submicron Microbial Cells and Viruses from Environmental and Biological Samples

Abstract This protocol set focuses on the preparation of the BD FACSAria II/III/Fusion, a cuvette‐based cell sorting system commonly found in shared resource settings, to sort submicron samples, including but not limited to virus‐like particles (VLPs) and bacteria. This is meant to serve as a proven workflow for staff in general shared resource laboratories (SRL) and individual labs. It is also useful for labs purchasing cuvette‐based sorters with similar fluidic paths to the FACSAria Fusion from BD Biosciences, such as the BD FACSSymphony S6 and BD FACSDiscover S8, as well as for specialized SRLs that will need to move away from Influx and MoFlo platforms that are approaching end of life. VLPs and submicron‐sized cells (e.g., ultramicrobacteria and archaea) are found at or near the limit of detection of most flow cytometers and cell sorters. With VLPs, the small quantity of DNA recovered requires amplification before downstream sequencing. Thorough cleaning of the fluidic system and careful sample preparation are necessary both to improve detection and to prevent genomic contamination from amplifiable free DNA or microorganisms. These protocols include instructions for the preparation of sheath fluid, decontamination of the cell sorter, and removal from the fluidic system of free DNA and endotoxin that could interfere with the high‐throughput amplification and sequencing of the target DNA in downstream processes. To minimize noise when sorting submicron‐sized samples, clean PBS filtered with a 0.1‐µm‐pore‐size filter is prepared, minimizing microbubbles and particulates; use of commercially available sheath fluids is not recommended, as they contain preservatives and surfactants that can affect microbial viability and are not filtered at the optimal pore size for this experimentation. In addition, detailed steps are provided for cleaning the instrument, tanks, and related media to prepare the sort, along with guidance for setting up the software, voltages, and gating strategies for successful experiments. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Preparation of the BD FACSAria II/III/Fusion cell sorter fluidics and software to perform sorts for validation by culture or microscopy Basic Protocol 2 : Preparation of the BD FACSAria II/III/Fusion cell sorter fluidics and software to perform sorts for high‐throughput whole‐genome amplification and genomic sequencing Support Protocol 1 : Autoclaving the BD FACSAria II/III/Fusion stainless‐steel sheath tank Support Protocol 2 : Chemical decontamination and maintenance of the BD FACSAria II/III/Fusion stainless‐steel sheath fluid tank Support Protocol 3 : Preparation of 1 L of 1× PBS Support Protocol 4 : Inspection of the BD FACSAria II/III/Fusion cell sorter for contamination Support Protocol 5 : Manual aseptic sorting procedure using BD FACSAria II/III/Fusion cell sorter Support Protocol 6 : Chemical decontamination and maintenance of the BD FACSAria II/III/Fusion wet cart containers Support Protocol 7 : Preparation of 1× PBS/0.1% (v/v) Tween 20 (PBST) Support Protocol 8 : Preparation of DNA‐free liquids and solutions Support Protocol 9 : Cleaning of the BD FACSAria II/III/Fusion nozzle by sonication