Quantification of algal cells using the Guava flow cytometer.

<p>The instrument was operated as described in Materials and Methods; all anemones were of strain CC7. (a) Effects of sample type and preparation method on the flow-cytometer plots of the red (chlorophyll) fluorescence vs. the side-scatter of the particles. The square within each plot indicates the region containing <i>Symbiodinium</i> cells with approximately normal chlorophyll concentration and light scatter. The samples of cultured algae (strain SSA02), fixed anemone homogenate, and coral homogenate (panels 1, 5, and 6) were prepared as described in Materials and Methods. The homogenates of aposymbiotic (APO) and symbiotic (SYM) anemones (panels 2 and 3) were prepared using our standard protocol except (i) the anemones had not been frozen and (ii) the symbiotic anemone was initially homogenized by rotor stator in ASW, after which one-fifth volume of 0.1% SDS in ASW was added before needle shearing and further dilution in 0.1% SDS in dH₂O. The frozen anemones (panel 4) were frozen in 0.1% SDS in ASW, then thawed, homogenized by rotor stator and needle shearing, and diluted further in the same solution. (b and c) Precision of the method and its ability to detect small differences in algal-cell concentrations. Anemone homogenates were prepared and diluted using our standard protocol. (b) Undiluted and diluted homogenates were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#pone.0135725.g003" target="_blank">Fig 3g</a> except that n = 8 in this case. (c) Serial two-fold dilutions of the homogenate were prepared, and the predicted (solid line) and measured (as mean values ± SEMs; n = 3) counts were compared. Most error bars are hidden by the points themselves with the graphing program used here. (d) Efficiency of automatic mixing by the instrument. Anemone homogenate was prepared by our standard protocol except that 0.1% SDS in ASW was used for homogenization and dilution. Samples were added to all wells of a 96-well plate, which was then analyzed in the usual way, so that individual samples sat for up to 84 min without agitation except for the automatic mixing that preceded the sampling from each well. R2 for the regression shown = 0.04. (e) Quantification of algal cells in two coral nubbins that had been held at 26°C and in two nubbins that had been stressed at 33°C for 3 d (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#sec002" target="_blank">Materials and Methods</a>). Homogenates were prepared as described in Materials and Methods, and algal cell numbers (see a, panel 6) were normalized to protein concentrations as determined by the BCA assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#sec002" target="_blank">Materials and Methods</a>).