Measuring coral host ash-free dry weight

Protocols used

• Putnam Lab AFDW Protocol

I followed the above protocol to quantify coral host ash-free dry weight of Pocillopora acuta (Pacu), Montipora capitata (Mcap) and Porites compressa (Pcom) for the ENCORE Hawaii TPC project.

Coral homogenate prep can be found in my previous post.

Detailed Protocol

1. Materials
   • Aluminum pans
   • Drying oven (60°C)
   • Muffle furnace (450°C)
   • Analytical balance (0.0001 g)
   • 15 mL falcon tubes
   • 5 mL pipet and tips
   • Centrifuge with rotors for 15 mL falcon tubes

2. Protocol

   1. Obtain aluminum weigh pans to be used in AFDW determination
   2. Label each pan with an ID number. May use a spatula to scrape a # into bottom of pan, or use pencil. Careful not to puncture a hole in the pan. Marker WILL burn off! Numbers written on the bottom of the pan will appear backwards and/or upside down when recording pan ID later on in protocol.
   3. After labeling, burn in the muffle furnace at 450°C for 4-6 h. After, remove from the muffle furnace, place in a desiccator for storage.
      Take care after this point that pans are not touched without gloves on, and that pans are ALWAYS sat on burned aluminum foil on tabletops and in ovens/furnaces/scales
   4. Record weight of burned pans on 4-decimal place scale (= “C” in Table 1.) Make sure you are using clean gloves or tweezers to weight the pans.
   5. Keep pans in desiccator until used.
   6. Remove frozen tissue homogenate from freezer and thaw.
   7. Vortex tissue homogenate and pipet 5 mL (use 5mL pipet) into a 15-mL falcon tube.
   8. Centrifuge the 15-mL tubes for 3 min at 3,500 xg.
   9. Line cafeteria trays with aluminum foil and fill with empty pre-burned pans, using tweezers to transfer pans.
   10. After centrifuging 15-mL tubes, pipet 4 mL of supernatant (host fraction) into a pre-burned pan. Record the pan number used for each sample, and indicate in notebook that this is the host fraction for that sample.
   11. Transfer trays of filled pans to drying oven at 60°C for at least 24 h.
   12. Weigh dried pans (using tweezers when transferring). Should be “at constant weight”. Leaving samples in drying oven longer than 24 h may be necessary, and is likely not detrimental.
   13. After samples have reached a constant weight, weigh pan + dried samples on a 4- decimal scale and record weight (= “D” in Table 1.)
   14. After recording the weight of the burned pan + dry tissue (and salts), place in the muffle furnace at 450°C for 4-6 h. Turn on the muffle furnace and click the ‘up’ arrow to set the temperature to 450 C. Once set, the muffle furnace will begin to heat up. After the 4-6 h time period, turn off the muffle furnace. This needs time (could be hours) for the muffle furnace to cool down. Budget for this in lab time. You cannot take the pans out right away. NOTE: The organic fraction will burn off at 450°C leaving only salt and inorganics behind. The difference between the dry weight and burned weight is the organic fraction of biomass.
   15. Let pans cool and transport to the scale room. Avoid transporting when warm, it will cause water to adhere to pans.
   16. Measure weight of burned pan + burned tissue (“D” – “F” in Table 1.). This is the AFDW of the organic fraction
   17. The AFDW will be biomass (g) for each mL of tissue added, which will then be normalized by the total homogenate volume and skeletal surface area.
   18. Table

   Table 1. Example calculations:

1. References

   1. Fitt et al., 2000. Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching. Limnol. Oceanogr., 45(3), 2000, 677–685
   2. Schoepf et al., 2013. Coral Energy Reserves and Calcification in a High-CO2 World at Two Temperatures. PLoS ONE 8: e75049
   3. Wall, C. 2015. Ash-free dry weight biomass assay protocol. Dr. Ruth Gates’ Laboratory Hawaii Institute of Marine Biology, University of Hawaii.