Multisizer 3 particle counter

Strain ploidy level was assessed using SYTOX® Green (Invitrogen) fluorescence and flow cytometry according to Delobel and Tesnière⁶⁵ , using an Accuri C6^TM flow cytometer. A single colony was selected and grown overnight in liquid YEPD with shaking at 150 rpm. Cells in exponential phase were collected by centrifugation and then re-suspended in 1 mL water. Eight ml of 75% ethanol were added and fixation was performed overnight at 4 °C. Fixed cells were harvested by centrifugation and washed with 1 mL PBS 1X. Cells were re-suspended in 500 μL RNAse A (Qiagen, Paris, France) solution (2 mg/mL RNAse A in 10 mM Tris-HCl, 15 mM NaCl) and incubated at 37 °C for 1 h and finally re-suspended in 200 μL of proteinase K (Roche, Paris, France) solution (1 mg/Ml in PBS 1X). After a 1 h- incubation at 50 °C, cells were collected by centrifugation, re-suspended in 500 μL PBS 1X, sonicated for 15 s, and stored on ice until analysis. Fifty µL of cell suspension were added to 200 μl of SYTOX® Green solution (1.25 µM SYTOX® Green in PBS 1X). Analysis was performed with a Multisizer 3 Particle Counter (Beckman Coulter, Paris, France). The FL1 detector (530/30 nm) was used for the acquisition of SYTOX® Green fluorescence, as this dye is taken up by cells in a manner stoichiometric to the amount of nuclear DNA.

Arizona test dust (ISO 12103-1) is used to make the PM test samples. The average diameters of PM_f and PM_c are 1.256 ± 1.309 µm and 7.657 ± 1.286 µm, respectively. The size distributions of PMs are measured by using the Multisizer 3 particle counter (Beckman Coulter, USA). The polydispersity index (PDI) is defined as the square of mean diameter of PMs divided by their standard deviation. The PDI values of PM_f and PM_c are 1.086 and 0.0282, respectively. Two reference samples of PM_f and PM_c with mass concentration values of 20 µg/ml are prepared by mixing PMs and distilled water. 20 mg of PM is measured by using an electronic balance (CAS, Korea) and then mixed with 1L of distilled water in a 1L wide neck bottle (Azlon, UK) to make two reference samples of PM_f and PM_c. The two reference samples and distilled water are then mixed in 30 ml square-shaped transparent bottles (Triforest, USA) at different volume ratios using 10 ml sterile syringes (Shinchang Medical, Korea) to prepare PM test samples with various concentration ratios of PM_f and PM_c.

Cell abundances in the cultures were determined with the use of epifluorescence microscopy (Carl Zeiss AxioScope.A1). Glutaraldehyde-fixed samples were stained with the nucleic acid dye DAPI (4′,6-diamidino-2-phenylindole, 0.03 M, Sigma-Aldrich), and filtered onto gray 0.2 µm pore-size polycarbonate membranes (GVS Life Technologies, ME). Chlorophyll-a emission by M. polymorphus cells and DAPI-stained bacteria were visualized under 450–490 nm excitation and 380–400 nm, respectively.
Volume concentrations of micro-aggregates “suspended” in cultures (i.e., non-sinking particles with an equivalent spherical diameter [ESD] of 5–60 µm) were determined at every sampling period using a Multisizer 3 Particle Counter (Beckman Coulter, CA). Prior to fixation with glutaraldehyde, duplicate samples were diluted to a 1–10% final particle concentration with Isoton II diluent (Beckman Coulter, CA) and aggregates were measured and quantified with a 100 µm aperture tube. The volume concentration of aggregates was calculated in µm³ per mL.