Computational protocol: High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

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Protocol publication

[…] To configure the flow cytometer, rinsed amoebae suspended in PAS were treated in three ways. First, at a concentration of 5 × 105 amoeba/ml, amoebae were mechanically lysed by sonication. Sonication causes the amoeba to burst under the effect of ultrasound delivered by a sterile probe plugged into the falcon tube containing the amoebae. We delivered a 60-Hz frequency for 1 min. The procedure was repeated four times. These mechanically treated amoebae served as our positive control for lysis. We infected the second suspension of amoebae, at the same concentration, with Mimivirus. The third suspension of amoebae remained intact and served as a negative control for lysis. The three specimens were then dispensed to a 24-well plate at 500 μl/well. After repeatedly pipetting to detach the adherent amoebae, a 250-μl fraction of each specimen was fixed in paraformaldehyde (4%) then transferred to a suitable tube for flow cytometric analysis on the BD LSRFortessa (BD Biosciences) at 0, 24, and 48 h after infection. Data acquisition was performed using log scales for instrument scatter parameters, forward scatter (FSC) and side scatter (SSC), respectively associated with size and internal complexity of the event analyzed. Protocol threshold was adjusted on FSC parameter. Acquisition and analysis were performed using “BD FACSDiva Software.” The number of collected events was fixed for all specimens. We gated our amoeba population using the density plot representation SSC versus FSC. We performed serial dilutions from 106 to 101 amoebae/ml to calibrate the analyzer, to determine the reliability of the counts and to establish the limit sensitivity of detection of the flow cytometer to choose a lysis threshold that corresponds to the loss of amoebae. All amoebal counts performed by flow cytometry were also performed on kova slides to validate the counts. The quantification was also verified using counting beads (true count or cytocount “DakoCytomation,” a suspension of concentration-calibrated fluorescent microspheres). This provides the absolute count of the amoebal population using the following equation: (number of cells counted/number of CytocountTM beads counted) × CytocountTM concentration (1100 beads/μl) × dilution factor (). We also used the Sytox nucleic acid stain for flow cytometry (Molecular Probes, Life Technologies, USA) to determine the viability of our host cells. All experiments were performed in triplicate. [...] By blind culture, without microscopic observation, we tested several samples of water and soil. Some of the samples were artificially contaminated with giant virus suspensions using Mimivirus and Marseillevirus (10 μl of virus at a concentration of 105 virus/ml were used to contaminate 3 ml of the sample). We contaminated 5 of 20 samples before proceeding to co-culture then for flow cytometry detection.In a second step, we tested 80 environmental samples, including 78 samples, previously tested negative by co-culture with A. polyphaga and the two samples from which the Terra1 and Terra2 Mimivirus strains had previously been isolated (). The processing steps for isolating these Terra1 and Terra2 strains have previously been described (; ). For our technique, we conducted three blind co-cultures as an enrichment step; primo-culture, sub-culture, and final culture without any microscopic observation. Data acquisition on the flow cytometer was performed 24 and 48 h following the final enrichment step. The number of collected events was fixed for all samples. Uninfected amoebae were used as a negative control. They were analyzed first and used to gate the population of amoebae according to the parameters defined above. The percentage of live and dead cells was evaluated. Moreover, the presence and percentage of debris characterizing amoebal lysis was also evaluated in the FSC SSC plot. All data from LSR Fortessa were analyzed using kaluza software (Beckman Coulter). An arbitrary threshold for amoebal loss of more than 50% after 24–48 h was used to define the possible presence of a lytic agent. […]

Pipeline specifications

Software tools BD FACSDiva, Kaluza
Application Flow cytometry