Putnam Lab Chlorophyll-A Concentration Protocol

Draft Protocol

Goal: To determine the concentration of Chlorophyll-A in adult coral tissue homogenate samples.

Protocol written for Montipora capitata and Pocillopora acuta adult coral samples from the Holobiont Integration 2018 project. Modified from Wall et al 2018.

Sample Preparation

1. Thaw homogenate aliquot labeled with the coral ID and “chlorophyll-a”.
2. Vortex to re-suspend the symbiont cell pellet.
3. Label a new microcentrifuge tube with the coral ID, total protein assay date, and “Chl-A”. If aliquot tube already contains exactly 500 μL of tissue sample, no need to make a new microcentrifuge tube.
4. Pipette 500 μL of the adult coral tissue sample into the new labeled 1.5 mL microcentrifuge tube.

Measuring Chlorophyll-A Concentration

1. Centrifuge the 500 μL aliquot of adult homogenate at 13,000 rpm for 3 minutes to separate the host and Symbiodiniaceae cells.
2. Remove the supernatant and discard.
3. Add 1 mL of 100% acetone to the 1.5 mL microcentrifuge tube.
4. Place in the dark at 4°C for 36 hours.
5. Measure this extracted chlorophyll-a pigment on the spectrophotometer at 630, 663, and 750 nm on a 96-well quartz plate.

Calculating Chlorophyll-A Concentration

Chlorophyll-A concentrations are calculated from the equations in Jeffrey and Humphrey 1975.
R code for this analysis can be found at K. Wong’s Github.