Growth of a common planktonic diatom quantified using solid medium culturing

Abstract

The ability to grow on solid culture medium is a pre-requisite for a successful microbial genetic model organism. Skeletonema marinoi, a bloom-forming, planktonic marine microalga, is widely used in ecological, evolutionary and population genetics studies. We have tested and confirmed the ability of this common organism to grow on solid culture medium (agar) under experimentally manipulated conditions. We established a protocol for quantifying growth characteristics - length of lag phase, growth rate, maximum biomass yield - on agar medium. The procedure was tested under experimental treatments and the resulting growth changes correlated with those observed in standard liquid culture. The ability to grow on solid medium broadens the use of S. marinoi as a molecular model, where agar is routinely used for various purposes (growth, selection, storage); and the possibility to quantify colony growth opens the way for high throughput, automated, or semi-automated phenotyping solutions.

Figure 1

Colony and cell-chain morphology of agar-cultured S. marinoi. The appearance of S. marinoi colonies on a surface of an agar-based solid medium, plated in quadruplicates in four 10-fold serial dilution steps (40–40 000 cells per colony), after one week on culturing at 10 °C (26 practical salinity units (psu); light intensity 50 µmol photons m⁻² s⁻¹) (a). The morphological appearance of S. marinoi (strain RO5AC) cells and chains under 400× magnification of an inverted light microscope after one week of culturing on solid medium (b), or standard liquid medium (c).

Figure 2

Graphical representation of a colony in three dimensions. z-axis represents the intensity values for each pixel within the colony area (x- and y-axis). The insert image shows the appearance of the corresponding S. marinoi colony in the exponential (a) and in the stationary (b) growth phases.

Figure 3

Quantitative phenotypes in liquid culture medium. Growth responses of three S. marinoi strains (GF0410J, HakH, RO5AC) to the treatment conditions (salinity 10 psu vs. 26 psu–x-axis; temperature 10 °C vs. 16 °C-red and blue boxes) in liquid f/2 + Si medium: maximum yield (ln-transformed chlorophyll a fluorescence values, a), growth rate (b), lag (days, c) (n = 4).

Figure 4

Quantitative phenotypes on solid culture medium (agar). Growth responses of three S. marinoi strains (GF0410J, HakH, RO5AC) to the treatment conditions (salinity 10 psu vs. 26 psu - x-axis; temperature 10 °C vs. 16 °C- red and blue boxes) on agar f/2 + Si medium: maximum yield (ln-transformed mean pixel values, a), growth rate (b), lag (days, c) (n = 32*). The results for both replicate plates are shown. * - the actual number of replicates used in the analysis was lower than 32 for some of the treatment plates due to the removal of dead colonies, see Methods: Statistical analysis.

Figure 5

Generalized treatment effects on growth phenotypes. Treatment effects on yield, rate, and lag are summarized into one value per phenotype by taking the mean of the corresponding values for all strains. The bars represent the ratio between these means for the two modes of the treatment (e.g. 10 °C and 16 °C for the temperature treatment). Phenotype ratios in salinity treatment (a), and in temperature treatment (b). Blue bars - liquid culture conditions, red bars - agar culture conditions. Error bars represent standard deviation from the mean for each group of values (n = 6).

Figure 6

Relationship between a phenotype estimate and its associated variance. Mean (n = 32*) strain and treatment-wise yield (ln-transformed mean pixel values, a), or growth rate (b) values (y-axis) are plotted against the corresponding variance values (x-axis). Linear regression trend line and the corresponding correlation coefficient (r2) are given for the RO5AC strain alone (dashed line) and for the whole data set (solid line). * - the actual number of replicates used in the analysis was lower than 32 for some of the treatment plates due to the removal of dead colonies, see Methods: Statistical analysis.

Figure 7

S. marinoi growth curves on replicate agar plates. Each curve represents an average smoothed time series of one of the three S. marinoi strains (GF0410J - red, HakH - green, RO5AC - blue) under the treatment conditions on agar medium supplemented with f/2+ Si medium. y-axis shows mean pixel values of each colony averaged across within-plate strain replicates (n = 32*). * - the actual number of replicates used in the analysis was lower than 32 for some of the treatment plates due to the removal of dead colonies, see Methods: Statistical analysis.