Ured using a MACAM UV203 radiometer (MACAM Photometrics, Livingston, UK) and together with the Ocean Optics SMS 500 spectroradiometer described above. Spectra have been measured in the variety 250?00 nm. All light measurements had been carried out inside the cylinders when they were wrapped with all the proper 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 development price (r) was calculated because the number of cell doublings day? by fitting an exponential function towards the logarithmic phase in the growth curve. Cell death was estimated working with SYTOX Green (Invitrogen, OR, USA) as outlined by the technique of Segovia and Berges (2009). Generally, 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 within the dark for 30 min and analysed by flow cytometry working with a DakoCytomation flow cytometer (MoFlo, Beckman Coulter, Fullerton, CA, USA) and under 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 had been analysed in triplicate. In vivo chlorophyll a fluorescence The optimal quantum yield of PSII fluorescence (Fv/Fm) was measured with a Water-PAM Ceftazidime (pentahydrate) Formula fluorometer (Waltz, Effeltrich, Germany) as described by Schreiber et al. (1986), thinking about Fv/Fm as (Fm ?Fo)/Fm in accordance with Genty et al. (1989), Fv is definitely the maximal variable fluorescence of a dark-adapted sample, Fm would be the maximal fluorescence intensity with all PSII reaction centres closed, and Fo is definitely the basal fluorescence. Higher Fv/Fm values indicate that cells are within a great situation, whereas a lower in Fv/Fm indicates strain and photoinhibition. Flow cytometry DAPI is really a popular nuclear counter-stain for use in multicolour fluorescent tactics. Its blue fluorescence stands out in vivid contrast to 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 as outlined by the method of Jim ez et al. (2009). Samples had been analysed utilizing a DakoCytomation flow cytometer. Counts had been triggered applying forward scatter signals. DAPI fluorescence was observed by way of a 435?85 nm band-pass filter and chlorophyll fluorescence via a 650?10 nm band-pass filter. Transmission electron LAU159 medchemexpress microscopy (TEM) Cells were harvested by centrifugation (15 min at 7000 g) and fixed in cacodylate buffer (100 mM, pH 7.two) containing four glutaraldehyde and 8.6 sucrose. Pellets were washed in 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. Right after dehydration in an ascending series of ethanol (70?00 ), samples have been embedded in four agar resin and ultrathin sections (60 nm thickness) were ready using 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 various magnifications. Quantification of cells by TEM can be problematic; for that reason, counting of cells displaying every single in the different characteristics was carried out for 3 fiel.