Full details of the experimental design are in:
Schoepf V, Grottoli AG, Warner ME, Cai W-J, Melman TF, Pettay DT, Hoadley K, Matsui Y, Baumann JH, Wang Y, Xu H, Li Q, & Hu X. 2013. Coral energy reserves and calcification in a high-CO2 world at two temperatures. PLoS ONE, 8(10): e75049. doi:10.1371/journal.pone.0075049.

A brief description of the analytical methods follows.

Coral collection (April 2011): northwest Fiji (17°29'19"S, 177°23'39"E)
Experiment (July/August 2011): Reef Systems Coral Farm, New Albany, Ohio, USA (40°07'24"N, 82°46'55"W)

Calcification. Net calcification was determined using the buoyant weight technique (Jokiel et al. 1978). Each coral fragment was buoyantly weighed at the beginning, middle (after 11 experimental days), and at the end of the experiment (after 23 experimental days). As such, it was possible to assess if calcification rates varied during the experiment. Daily calcification rates were calculated as the difference between initial, middle, and final weights, divided by the respective number of days elapsed, and standardized to surface area (see below).

For tissue analyses, corals were frozen at −80 degrees C and a total of three branch tips or growing edge pieces were saved from each fragment for lipid, protein/carbohydrate, and tissue biomass analyses, respectively. The remaining tissue was airbrushed for chlorophyll a and endosymbiont density measurements.

Chlorophyll a and endosymbiont density. Coral tissue was stripped off the coral skeleton with a waterpik containing 40 ml of synthetic seawater (Instant Ocean). The endosymbionts were isolated from the host tissue via centrifugation and then resuspended in 10 ml of synthetic seawater. For chlorophyll a concentrations, 1 ml of this algal suspension was pelleted and the cells lysed in 1 ml of 4 degree C methanol using a bead-beater for 60 seconds. Samples were then immediately placed on ice and allowed to extract for one hour in the dark. Samples were centrifuged to remove cellular debris and measured spectrophotometrically (λ = 652, 665, & 750) on a 96-well plate reader. The equations for chlorophyll a in methanol described by Porra et al. 1989, along with path length correction (Warren 2008), were used to calculate chlorophyll a concentrations (pg/cell), and were then standardized to surface area (see below). Another 1 ml subsample of the algal suspension was preserved with 10 ul of 1% glutaraldehyde solution for endosymbiont quantification, which was calculated using 6 independent replicate counts on a hemocytometer, using a Nikon microphot-FXA epifluorescent microscope at 100× magnification. Photographs were analyzed through Image J using the analyze particles function.

Energy reserves and tissue biomass. For all energy reserve and tissue biomass measurements, only branch tips or samples with a growing edge were used. While tissue composition may vary across the surface of a coral (Oku et al. 2002), this approach was used to allow for comparison with previously published studies (Grottoli et al. 2004, Rodrigues and Grottoli 2007, Levas et al. 2013). Soluble lipids (referred to hereafter simply as lipids) were extracted from a whole, ground coral sample (skeleton + animal tissue + algal endosymbiont) in a 2:1 chloroform:methanol solution for 1 hour (Grottoli et al. 2004, Rodrigues and Grottoli 2007), washed in 0.88% KCl followed by 100% chloroform and another wash with 0.88% KCl. The extract was dried to constant weight under a stream of pure nitrogen (UPH grade 5.0) and standardized to the ash-free dry weight.

Animal soluble protein and carbohydrate (referred to hereafter simply as protein and carbohydrate, respectively) were extracted from grinding a whole second branch tip of the same fragment (Rodrigues and Grottoli 2007). Briefly, Milli-Q water was added to the ground coral sample and the resulting slurry was sonicated (5 min) and then centrifuged twice (5000 rpm, 10 min) to separate the animal tissue from the skeleton and endosymbiotic algae. Protein and carbohydrate was extracted from the animal tissue only. One subsample of this animal tissue slurry was used for protein extraction using the bicinchoninic acid method (Smith et al. 1985) with bovine serum albumin as a standard (Pierce BCA Protein Assay Kit). A second subsample was used for carbohydrate quantification using the phenol-sulfuric acid method (Dubois et al. 1956) with glucose as a standard. Soluble animal protein and carbohydrate concentrations were standardized to the ash-free dry weight.

Tissue biomass was measured by drying a third branch tip of whole coral sample (skeleton + animal tissue + algal endosymbiont) to constant dry weight (24 hours, 60 degrees C) and burning it (6 hours, 450 degrees C). The difference between dry and burned weight was the ash free dry weight which was standardized to the surface area of this branch tip.

Surface area. Surface area of plating M. monasteriata and T. reniformis fragments was determined using the aluminum foil technique (Marsh 1970), whereas surface area of branching A. millepora and P. damicornis fragments was determined using the single wax dipping technique (Stimson and Kinzie 1990, Veal et al. 2010) after the tissue had been removed. Natural wooden blocks of varying sizes and shapes were used as calibration standards (Veal et al. 2010). Wax dipping was conducted using household paraffin wax (Gulf Wax, Royal Oak Enterprises) heated to 65 degrees C. Dried coral skeletons and wooden calibration standards were maintained at room temperature prior to weighing.