Goal: Determine ash-free dry weight in Astrangia samples from 2021 experiment

This protocol was adapted from the Putnam lab.

Materials & Equipment

• Aluminum pans
• Drying oven (60°C)
• Muffle furnance (450°C)
• Analytical balance (0.0001)
• 15 mL falcon tubes
• 5 mL tubes
• P1000 + tips
• 1x PSB
• Centrifuge for 15 mL falcon tubes
• Desiccator

Samples should be aliquoted from homogenate generated post-airbrushing. See airbrushing protocol here.

Protocol

1. Obtain aluminum weigh pans for AFDW
2. Label each pan with an ID number. Use a spatula or a pencil to scrape a number into the bottom of the pans
   • Careful not to puncture a hole in the pans
   • Do not use marker, as it will burn off
   • Keep in mind that numbers written on the bottom of the pan will appear backwards/upside down
3. After labeling, burn the pans in the muffle furnance at 450°C for 4-6 hours
   • Make sure pans are not touched without gloves on and that pans always sit on burned aluminum foil when on the tabletop
4. Record weight of burned pans on analytical balance (to 4 decimal places)
   • Record under Tin_g
5. Keep pans in desiccator until used
6. Thaw frozen homogenate (should be in 15 mL tubes labeled AFDW)
7. Centrifuge the 15 mL tubes for 5 min at 3,500 rpm
8. Aliquot 4 mL of the supernatent (host) into 5 mL tube. Pour out the rest of the supernatent into a waste jar
9. Add 1 mL of 1x PBS to the pellet (symbiont). Pipette up and down to mix.
10. Keep host and symbiont fractions in -80°C until ready to burn
11. Once ready to burn, pipet 4 mL of the host fraction into a pre-burned pan. Record the pan number used for each sample and record that this is the host fraction.
12. Pipette 1 mL of the symbiont fraction into a pre-burned pan. Record the pan number used for each sample and record that this is the symbiont fraction.
13. Transfer the trays of filled pans to drying oven at 60°C for at least 24 hours
14. Weigh pans after 24 hours. They should be at a “constant weight”
    • May need to weigh pans at 20, 24, and 28 hours to ensure samples have reached a constant weight.
    • Record under Tin_Dry-g
15. After recording the weight of the burned pan + dried tissue, put the pans in the muffle furnance at 450°C for 4-6 hours
    • The organic fraction will burn off at 450°C leaving only salt and inorganics behind. The difference between the dry weight (Tin_Dry_g) and burned weight (Tin_Burn_g) is the organic fraction of biomass
16. Weigh pans after 4-6 hours
    • Record under Tin_Burn-g
17. Sample calculations are below

Calculations

References

• 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
• Schoepf et al., 2013. Coral Energy Reserves and Calcification in a High-CO2 World at Two Temperatures. PLoS ONE 8: e75049
• Wall, C. 2015. Ash-free dry weight biomass assay protocol. Dr. Ruth Gates’ Laboratory Hawaii Institute of Marine Biology, University of Hawaii.