Physiological Parameters Protocol Testing

Goal: To determine the minimum amount of homogenate volume necessary from healthy and bleached fragments to produce accurate readings from physiological parameters (total protein, chlorophyll-A concentration, lipid content, total antioxidant capacity, symbiont counts).

20191023 E.S. Airbrushing, Homogenization, Aliquoting, Bleaching

Two healthy and two bleached fragments were chosen from the 20181215 molecular back-burner fragments and airbrushed/homogenized following the Putnam Lab Airbrush protocol.

Coral ID	Tank	Treatment	Timepoint	Airbrush_Date	Tech	Homogenate_volume_mL	Notes
M1567	9	HTAC	20181215	20191023	ES	20	Bleached
P1615	4	HTAC	20181215	20191023	ES	17	Bleached
M2193	11	ATHC	20181215	20191023	ES	30	Healthy
P2738	11	ATHC	20181215	20191023	ES	15	Healthy

Coral skeletons were placed in 20% bleach for 24 hours (20191023), and then left out to dry for 24 hours (20191024).

20191030 E.S./S.G.
Total Antioxidant Capacity following this protocol was tested on the four above coral fragment samples.

Coral ID	Timepoint	Well ID	Pre_Reaction	Post_Reaction
1567_1	20181215	B09	0.043	0.089
1615_1	20181215	B10	0.044	0.11
2193_1	20181215	B11	0.043	0.093
2738_1	20181215	B12	0.049	0.11
1567_2	20181215	D1	0.043	0.1
1615_2	20181215	D2	0.045	0.126
2193_2	20181215	D3	0.043	0.097
2738_2	20181215	D4	0.05	0.128

20191031, 20191101 E.S.
Ash-Free Dry Weight following this protocol was tested on the four above coral fragment samples. 5 volumes (100 μL, 250 μL, 400 μL, 600 μL, 700 μL) were tested for each fragment to determine the minimum volume needed for a signal for both healthy and bleached fragments.

Coral ID	Timepoint	Initial_weight	Post_oven	Post_furnace	AFDW_Date	AFDW_Tech	Notes
1615_100	20181215	0.0937			20191031	ES	Protocol testing
1615_250	20181215	0.2431			20191031	ES	Protocol testing
1615_400	20181215	0.3816			20191031	ES	Protocol testing
1615_600	20181215	0.5823			20191031	ES	Protocol testing
1615_700	20181215	0.6919			20191031	ES	Protocol testing
1567_100	20181215	0.1015			20191031	ES	Protocol testing
1567_200	20181215	0.2316			20191031	ES	Protocol testing
1567_400	20181215	0.3902			20191031	ES	Protocol testing
1567_600	20181215	0.5859			20191031	ES	Protocol testing
1567_700	20181215	0.6793			20191031	ES	Protocol testing
2193_100	20181215	0.0948			20191031	ES	Protocol testing
2193_250	20181215	0.2383			20191031	ES	Protocol testing
2193_400	20181215	0.3812			20191031	ES	Protocol testing
2193_600	20181215	0.5852			20191031	ES	Protocol testing
2193_700	20181215	0.6767			20191031	ES	Protocol testing
2738_100	20181215	0.0964			20191031	ES	Protocol testing
2738_250	20181215	0.236			20191031	ES	Protocol testing
2738_400	20181215	0.3847			20191031	ES	Protocol testing
2738_600	20181215	0.5808			20191031	ES	Protocol testing
2738_700	20181215	0.6902			20191031	ES	Protocol testing

20191104 E.S.
Total Protein following this protocol was tested on the four above coral fragment samples.

Notes:

• 280 μL was too much 0.1 M HCl, pH read 5.0
• 50 μL of 1 M NaOH added back, pH read 9.0
  Just to #1567:
• 10 μL HCl added, pH read 9.0
• 10 μL HCl added, pH read 9.0
• 10 μL HCl added, pH read 9.0
• 50 μL HCl added, pH read 9.0
• 50 μL HCl added, pH read 8.5
• 50 μL HCl added, pH read 8.5
• 50 μL HCl added, pH read 8.5
• 50 μL HCl added, pH read 7.0
  Just to #1615:
• 200 μL HCl added, then 50 μL HCl added, then 10 μL HCl added, then 10 μL HCl added until pH read 7.0
  Just to #2193:
• 200 μL HCl added, then 10 μL HCl added until pH read 7.0
  Just to #2738:
• 270 μL HCl added until pH read 7.0

Map for Spectrophotometer:

1	2	3	4	5	6	7	8	9	10	11	12
A	A	A	B	B	C	C	D	D	E	E	F	F
B	G	G	H	H	I	I	1567	1567	1615	1615	2193	2193
C	2738	2738
D

Total Protein Data Sheet