Ed the evaluation of algal cell composition, but did not permit

Ed the evaluation of algal cell composition, but did not permit a reliable evaluation of the specific growth rate. The grazers and their visible wastes (e.g. copepods fecal pellets) were separated from the culture by filtration prior to any measurement. In order to get rid of any residual wastes, further washes were effected with fresh medium and an ammonium formate solution isosmotic to the culture media. The impact of wastes that may have remained in the culture was also tested in preliminary checks and found to be negligible in all cases and for all measurements.Growth rate, cell size and dry weight, chlorophyll fluorescenceCell numbers and volumes were measured with automatic cell counter CASY TT (Innovatis AG, Retlingen, Germany; [12]). Specific growth rates, , were derived from daily counts of exponentially growing cells, carried out on a minimum of three distinct cultures for each treatment. In the experiments conducted in the presence of ciliates or copepods, grazer feeding rate was not considered; the growth rates are thus netPLOS ONE | www.plosone.orgEvolution of Phytoplankton-Grazers Interactionof grazing. For dry weight determination, cells were washed with an isosmotic ammonium formate solution and dried at 100 until weight stabilized. Measurements were conducted for at least three independent cultures. The chlorophyll fluorescence associated to PSII was studied using a Dual-PAM-100 fluorometer according to [5].AMPC The maximum fluorescence yield and the dark fluorescence yield of dark adapted (Fm and Fo, respectively) and illuminated (Fm’ and Fo’) cells were determined. The maximal quantum yield of PSII and the quenching parameters qP and NPQ were derived from these measurements according to [13,14].Praziquantel Data acquisition and analysis were conducted using the Dual-PAM v1.8 software (Walz GmbH, Effeltrich, Germany).Elemental compositionCell quotas of C, N and S were determined using an elemental analyzer (EA1108, Carlo Erba Instruments, Milan, Italy) as described in [5].PMID:23329319 One to six milligrams of cells (dry weight) were washed twice with an ammonium formate solution isosmotic to the culturing media and dried at 80 until the weight stabilized. The washes with ammonium formate (which is volatile at high temperature) were necessary to eliminate growth medium salts that would have interfered with the determination of dry weight and, consequently, cell stoichiometry. Sulphanilamide (C:N:S= 6:2:1) was used as a standard. Elemental quotas were calculated as pico- or femtograms per cell, normalized to cell dry weight and to cell volume. Data acquisition and analysis were performed with the software EAS-Clarity (DataApex Ltd. 2006, Czech Republic). All measurements were repeated for four independent cultures. The abundance of elements with an atomic mass between 24.305 g mol-1 (Mg) and 238.03 g mol-1 (U) was measured using a Total Reflectance X-ray Fluorescence spectrometer (S2 Picofox, Bruker AXS Microanalysis GmbH, Berlin Germany). Dry algae cultured at 5 mM or 30 mM SO42(1010-1011 cells L-1) were resuspended in 1 mL of dH2O and vortexed until the suspension was homogeneous. A solution of 1 g L-1 Ga (Sigma Aldrich, St. Luis, MO, USA) in 5 HNO3 was added as internal standard to a final concentration of 5 L L-1. Aliquots of 10 of this suspension were deposed on a quartz sample holder, dried on a heating plate for 10 minutes, and measured for 1000 seconds. Spectral deconvolution and quantification of elemental abundances were performed by.