Ured using a MACAM UV203 radiometer (MACAM Photometrics, Livingston, UK) and with the Ocean Optics SMS 500 spectroradiometer mentioned above. Spectra have been measured within the variety 250?00 nm. All light measurements had been carried out inside the cylinders as soon as they have been wrapped together with the suitable cut-off filters. Cell abundance and cell death For cell counts, 1 ml of fresh cell culture was counted within a Coulter Counter (Z2 Beckman Coulter, Fullerton, CA, USA). The growth rate (r) was calculated as the variety of cell doublings day? by fitting an exponential function to the logarithmic phase of your development curve. Cell death was estimated using SYTOX Green (Invitrogen, OR, USA) in accordance with the strategy of Segovia and Berges (2009). Basically, cell pellets have been resuspended in 1 ml of 10 mM PBS buffer (pH 7) containing SYTOX Green at a final concentration of 20 , incubated at 16 inside the dark for 30 min and analysed by flow cytometry applying a DakoCytomation flow cytometer (MoFlo, Beckman Coulter, Fullerton, CA, USA) and below an epifluorescence microscope (Leitz, Wetzlar, Germany) at an excitation wavelength of 450?90 nm and emission wavelength of 523 nm. Constructive controls consisted of cells killed by fixation with 1 glutaraldehyde. Samples were analysed in triplicate. In vivo chlorophyll a fluorescence The optimal quantum yield of PSII fluorescence (Fv/Fm) was measured using a Water-PAM fluorometer (Waltz, Effeltrich, Germany) as described by Schreiber et al. (1986), contemplating Fv/Fm as (Fm ?Fo)/Fm based on Genty et al. (1989), Fv could be the Anilofos Epigenetics maximal variable fluorescence of a dark-adapted sample, Fm is the maximal fluorescence intensity with all PSII reaction centres closed, and Fo would be the basal fluorescence. High Fv/Fm values indicate that cells are inside a very good situation, whereas a decrease in Fv/Fm indicates pressure and photoinhibition. Flow cytometry DAPI is actually a common nuclear counter-stain for use in multicolour fluorescent techniques. Its blue fluorescence stands out in vivid P3 Inhibitors targets contrast for the green,5262 Garc -G ez et al.yellow, or red fluorescent probes of other structures and it particularly stains nuclei, with small or no cytoplasmic labelling. DAPI (Molecular Probes, Eugene, OR, USA) was added at a concentration of 1?0 and incubated for 5 min at space temperature in line with the system of Jim ez et al. (2009). Samples had been analysed utilizing a DakoCytomation flow cytometer. Counts had been triggered making use of forward scatter signals. DAPI fluorescence was observed via a 435?85 nm band-pass filter and chlorophyll fluorescence via a 650?ten nm band-pass filter. Transmission electron microscopy (TEM) Cells were harvested by centrifugation (15 min at 7000 g) and fixed in cacodylate buffer (100 mM, pH 7.two) containing 4 glutaraldehyde and eight.6 sucrose. Pellets had been washed inside a series of cacodylate buffers with descending sucrose concentration and post-fixed in 1 osmium tetroxide dissolved in Milli-Q ultrapure water (Millipore, USA) for 2 h. Immediately after dehydration in an ascending series of ethanol (70?00 ), samples were embedded in four agar resin and ultrathin sections (60 nm thickness) had been prepared with a Reichert-Jung ultramicrotome (Leipzig, Germany). Sections have been stained with uranyl acetate and lead citrate, and observed under a Philips CM 100 transmission electron microscope at diverse magnifications. Quantification of cells by TEM might be problematic; consequently, counting of cells showing every single of your distinct traits was carried out for 3 fiel.