Protocol/Methods For First Bailout Experiment.

1. Assure the Setup is Correct. Bailout Setup
2. Turn on incubator 24hrs before test day allowing the header tank to get to 25 degrees Celsius
3. Assure that the AI Lights are on and on schedule.
4. Check the Salinity of the Header Tank once it is up to temperature (60-63ppt)
5. Create 8 clippings of Pocillopora Acuta using Bone Clippers. Clippings should be 1-2cm long with 20-25 polyps.
6. Take pictures of each clipping.
7. Add 25 degree 35ppt saltwater from the same tank as the clippings into each chamber C1-C8.
8. Place clippings gently into each chamber make sure they are not touching walls or bubbler tips.
9. Attach Bubble tips and Input tubes to C1-C4
10. Attach Bubble tips only to C5-C8 these will be control chambers salinity will stay constant.
11. Close all chambers and plug in air pump
12. Let run for 20 hours after 20 hours check hourly for signs of bailout. If they dont bailout use a small pipette to agitate the water around them.
13. Take pictures of coral skeleton after bailout.
14. Use dissecting microscope to determine the health of coral micropropogates.

1. Rinse all Chambers with DI water to make sure they are clean and dry.
2. Ensure that the header tank is at 60ppt or close to it within .5
3. Using a 25ml graduated cylinder add 23ml of 35ppt water to each test chamber.
4. Test the salinity of each chamber (we are using a Fisher Brand Salinity/Temp meter pen) Salinty Pen Link
5. Turn on Incubator so it can get to temp (25 degrees Celsius)
6. Run inlet Jebaos to assure that there are not a lot of bubbles or clogs in the lines.
7. plug inlet water airline and airline into the top of chambers ensure that there are bubble mixing the water.
8. Wait for first 1ml dose. 15minutes after the dose check the salinity of every chamber. Make sure air pump is turned off before checking the salinity.
9. Let Run for 24 hours checking salinity of every chamber every hour.
10. Rinse salinity probe end with DI water between measurements dry it with a KimWipe.
11. Enter hourly measurements into a .CSV file and run a linear regression for each chamber.

1. Hook up jebao fully with the correct length of airline tubing you will be using.
2. Enter calibration mode on the Jebao up arrow choose the pump you would like to calibrate
3. Normally when calibrating a jebao it will keep track of the time it takes for the pump to dispense 100ml of DI water into a graduated cylinder. It usually take 200-225 seconds.
4. Instead of dispensing 100ml only dispense 10ml using a smaller graduated cylinder. This will end up only being 18-25 seconds
5. Because you are only using 10ml to calibrate it, you need to make sure all your doses are changed accordingly. If you would like to Dose 1ml of fluid instead set the jebao to dose 10ml.
6. In changing the calibration you have made the jebao more fine tuned and it will pump out a more accurate 1ml then when set to 100ml calibration.

Salt Mixing with Reef Pro Mix

1. Scan QR Code on salt box. RPM Website
2. Enter the gallons of water you are mixing and your desired salinity.
3. I have been using 3 gallons of water to make 60ppt salt water
4. Weigh out the correct amount of salt with scale.
5. 
6. Use a small pump to mix water together.
7. mix for 12 hours adding salt if needed.

All of the setup is in a Thermo Scientific incubator set to 25 degrees Celsius

1. We used a organizing tray with 36 cells. Each cell has a height of 2.5cm a width of 3cm and a length of 4cm. -Should have enough space to hold 23ml of water. link for tray
2. I used 2 trays and used only the 4 front facing cells from each tray. For a total of 8 test chambers.
3. In each test chamber inlet holes where drilled in the top with a 6mm drill bit.
4. Air bubbler inlet holes where drill in the top with a 6mm drill bit.
5. Outlet holes drilled with a 5mm drill bit about 2/3 up the side wall. facing out. 6.AquaCulture Air Pump
6. Air bubbles used for mixing water. I attached a micro pipette tip to airline and put the tip in the chamber.
7. Air controlled with valves above every outlet tip. Valves Connectors
8. Jebao DP4s where used to dose 1ml of water in every hour and to take 1ml out of the chamber after 10 minutes. Jebao DP4
9. Aqua Illumination Hydra WiFi Led Lights where hung 28 cm above the chambers. AI Light
10. Calibrated to output 65-70 PAR. Below are the wavelength percentages.
11. Higher salinity water doses From a Header Tank at the bottom of the system.
12. Water that leaves the camber enters a waste water container via jebao pump.
13. All Air Line is 3/16. Airline used for both water and bubbler. Airline
14. Final Setup.

this is a test