Rapid, Precise, and Accurate Counts of Symbiodinium Cells Using the Guava Flow Cytometer, and a Comparison to Other Methods
Fig 3
Quantification of algal cells using the Coulter Counter.
All samples except those in f and h were diluted into Isoton before counting (see Materials and Methods). (a-f) Particle-size distributions. (a) A sample of cultured Symbiodinium strain SSA02 (Culture) was prepared as described in Materials and Methods, and homogenates of symbiotic (SYM) and aposymbiotic (APO) anemones were prepared using our standard protocol but with ASW rather than dH2O. (b) Fresh (i.e., not previously frozen) anemones were homogenized in ASW using the rotor stator, and samples were mixed one-to-one with either ASW or 10% SDS in ASW and needle-sheared. (c) Fresh anemones were homogenized in dH2O or ASW using the rotor stator, mixed with one-fifth volume of 1% SDS in dH2O or ASW, respectively, and needle-sheared. (d) Anemones were homogenized (rotor stator plus needle-shearing) in 0.1% SDS in ASW with or without prior freezing and thawing. (e) Anemone homogenates were prepared with or without formaldehyde fixation after homogenization as described in Materials and Methods. (f) Fresh anemones were homogenized in ASW containing 1% SDS using either the rotor stator or a manual tissue homogenizer (see Materials and Methods); the homogenates were diluted 20-fold with filtered ASW before counting. (g) A homogenate was prepared using our standard protocol, and the algal concentrations were measured in both an undiluted sample and a sample diluted to 95% of the original concentration. Gray bars, means of four replicate measurements; black lines, SEMs (too small to be resolved on this scale); dotted line, predicted 95% value. (h) A homogenate of fresh anemones was prepared in ASW using the manual tissue homogenizer (see f). One-tenth volume of 1% SDS in dH2O was added, the sample was diluted ~60-fold into ASW, and 10-ml aliquots were placed into two Coulter-Counter cuvettes. Each sample was mixed thoroughly and counted (t = 0). At 5-min intervals thereafter, each sample was counted again, in one case with no further mixing and in the other case with a thorough mixing prior to each count. Mixing was performed carefully to avoid introducing air bubbles. R2 values for the regressions shown are 0.001 (with mixing) and 0.94 (without mixing). In g and h, particles of 6.5–12 μm (cf. the plots in a-f) were included in the counts.