TITLE:  Oxygen Evolution Measurement: Light Curve for Algae 
AUTHORS: Steven J Burgess, Chandra   Davies

[DESCRIPTION]
Thanks to a lovely combination of trial and error I present to you an optimized* protocol for using an oxygen electrode to create a light curve for Ostrecoccus tauri. The light curve can be used to determine what light intensity is ideal for photosynthesis in O. tauri, which can then inform the light conditions for a subsequent bicarbonate curve. An O. tauri bicarb curve could potentially indicate (when compared to other algal bicarb curves) whether or not this species has a carbon-concentrating mechanism.

*This protocol could be further optimized by making a growth curve for O. tauri first. 

*The protocol is written for O. tauri and can be adapted to other algal species. When adapting it will be necessary to reassess optimal cell density for measurements, and consider the choice of buffer for washing and incubating cells. ASW is used in this situation but will not be suitable for fresh water species

[BEFORE_START]
Have a checklist - (1) water jacket,  (2) stirrer, (3) lid as a reminder to do these steps each time

[STEPS]
SECTION: Prepare the electrolyte
2. Prepare the electrolyte

SECTION: Prepare the electrolyte
2.2. Pour the salt into a 500 mL glass bottle

SECTION: Prepare the electrolyte
2.3. Measure 100 mL of dH2O

SECTION: Prepare the electrolyte
2.4. Pour the water into the same bottle as salt
SECTION: Prepare the electrolyte
2.5. Close the lid and swirl the bottle with mild aggression until salt is dissolved
SECTION: Prepare 0.5M NaHCO3 Solution
1. Prepare 0.5 M NaHCO3 Solution

SECTION: Prepare 0.5M NaHCO3 Solution
1.1. Dispense 10 mL of dH2O into a 50 mL centrifuge tube

SECTION: Prepare 0.5M NaHCO3 Solution
1.3. Add 0.420 g of sodium bicarbonate to the dH2O

SECTION: Prepare 0.5M NaHCO3 Solution
1.4. Close lid and invert until everything is dissolved
SECTION: Clean and Prepare the electrode
3.9. Cut a slightly larger square of the provided PTFE membrane tape and place it on top of the paper.
 
  

SECTION: Clean and Prepare the electrode
3.8. Cut a 1.5-2 cm2 piece of thin cigarette paper to the top of the electrode.

 

SECTION: Clean and Prepare the electrode
3.7. Add 3-5 drops of water so that the silver band inside the well is completely covered.
SECTION: Clean and Prepare the electrode
3.6. Use a disposable Pasteur pipette to add a drop of electrolyte to the top of the platinum electrode.
SECTION: Clean and Prepare the electrode
3.5. Use a clean, dH2O-dampened cotton bud to remove the residual polish. A kimwipe can be used to dry the electrode after this step, too.
SECTION: Clean and Prepare the electrode
3.4. Polish the platinum electrode as well. 
 

SECTION: Clean and Prepare the electrode
3.3. Circle the inside of the well with the polish
SECTION: Clean and Prepare the electrode
3.2. Dampen a cotton bud with dH2O and use it to pick up a thin layer of Hansatech Rapid Electrode Disc Polish to clean the electrode
SECTION: Clean and Prepare the electrode
3.1. Start the water jacket going at 25 oC so it’s ready by the time your electrode is prepared
SECTION: Clean and Prepare the electrode
3. Clean and Prepare the electrode
SECTION: Clean and Prepare the electrode
3.10. Use the membrane applicator to secure the small O-ring over the membrane and cigarette paper at the base of the electrode
  

SECTION: Clean and Prepare the electrode
3.11. Place the large O-ring in the indentation outside the well
 

SECTION: Clean and Prepare the electrode
3.12. Tuck any extra edges of membrane into the well using forceps so it doesn't touch the larger O-ring
  

SECTION: Clean and Prepare the electrode
3.13. Attach the cord that connects the control unit to the electrode, then fit the disc into the bottom of the electrode chamber (pictures in steps 4.1-4.2).




SECTION: Clean and Prepare the electrode
3.14. Open O2View program on laptop
SECTION: Clean and Prepare the electrode
3.15. Add port cover blanks to 3 of the open ports and insert the red light to the 4th open port.

   

SECTION: Clean and Prepare the electrode
3.16. Insert the light sensor to the top opening of the electrode chamber, ensuring the circular patch is facing the light source (pictures in steps 4.1-4.2).

SECTION: Light Calibration
4. Light Calibration
 

SECTION: Light Calibration
4.1. Ensure both the light and light sensor are inserted to their appropriate ports, and that the light sensor is actually facing the light
    

SECTION: Light Calibration
4.2. In the O2View program, in the upper menu bar, click Calibrate > calibrate light (automatic)
SECTION: Light Calibration
4.3. This should take a few minutes but it will do it itself.
SECTION: Electrode Calibration
5. Electrode calibration



SECTION: Electrode Calibration
5.1. Add 1 mL of ASW to the electrode chamber
 


SECTION: Electrode Calibration
5.2. Insert plunger (hollow lid-like contraption with glass on one end. Glass end goes into the chamber)
  

SECTION: Electrode Calibration
5.3. Calibrate > Liquid phase calibration (manual)
SECTION: Electrode Calibration
5.4. Follow the prompts that pop up on the screen
SECTION: Electrode Calibration
5.5. Use their default 25 μmol/mL oxygen concentration setting.
 
  


SECTION: Electrode Calibration
5.6. Enter stirrer speed of 75
  

SECTION: Electrode Calibration
5.7. Continue to follow prompts:
  
  

 

SECTION: Electrode Calibration
5.8. Nitrogen gas was introduced to the chamber from a large tank at the end of the bench through a tube with a 1000 μL pipette tip attached. The pipette tip goes directly into the ASW in the electrode chamber, preferably without jamming it into the electrode disc. Leave it in until the signal has plateaued and the calibration is saved.

  

SECTION: Prepare Light Curve Culture Samples
6. Preparing Light Curve Culture Samples

 

SECTION: Prepare Light Curve Culture Samples
6.1. In a flow hood, pour 25 mL of a healthy culture into a 50 mL centrifuge tube

 

SECTION: Prepare Light Curve Culture Samples
6.2. Prepare a balance with another tube of 25 mL of dH2O

SECTION: Prepare Light Curve Culture Samples
6.3. Centrifuge for 5 minat 5000 g and 20oC



SECTION: Prepare Light Curve Culture Samples
6.4. Discard the supernatant by micropipetting
SECTION: Prepare Light Curve Culture Samples
6.5. Resuspend in 10 mL of ASW by gently disturbing the pellet with a small paintbrush

 

SECTION: Prepare Light Curve Culture Samples
6.6. Balance with dH2O again in centrifuge for 5 min, 5000 g, 20 oC

SECTION: Prepare Light Curve Culture Samples
6.7. Discard the supernatant
SECTION: Prepare Light Curve Culture Samples
6.8. Add 1 mL ASW to the pellet

SECTION: Prepare Light Curve Culture Samples
6.9. Swirl the tube gently
SECTION: Prepare Light Curve Culture Samples
6.10. Use the paintbrush again to resuspend. Press the paintbrush against the inside of the tube when pulling it out to try and save as much of the sample as possible.
SECTION: PFD Table Set up for Light Curve
7. Photon Flux Density Table Set up for Light Cruve
SECTION: PFD Table Set up for Light Curve
7.1. Remove ASW from chamber
SECTION: PFD Table Set up for Light Curve
7.2. Make sure the stirrer is still going (can turn off while removing ASW)
SECTION: PFD Table Set up for Light Curve
7.3. In the top menu bar, click Hardware > New PFD table
SECTION: PFD Table Set up for Light Curve
7.4. Select auto
SECTION: PFD Table Set up for Light Curve
7.5. Adjust so the following intensities of light (umoles) are selected for 1 minute each: 0, 5, 20, 40, 80, 120, 200, 400, 800, 1600. Click save if you need to close the window, otherwise leave the window up until you clock "go."
  

 

SECTION: PFD Table Set up for Light Curve
7.6. Add 900 µL of culture

SECTION: PFD Table Set up for Light Curve
7.7. Add 100 µL of NaHCO3 solution

SECTION: PFD Table Set up for Light Curve
7.8. Place the plunger into the chamber
SECTION: PFD Table Set up for Light Curve
7.9. Click "Go" on PFD window
SECTION: PFD Table Set up for Light Curve
7.10. When program is done, turn off stirrer
SECTION: PFD Table Set up for Light Curve
7.11. View QY Data tab and save the file

 
 


SECTION: Clean Up
8. Clean Up
SECTION: Clean Up
8.2. Make sure everything is turned off (stirrer, light)
SECTION: Clean Up
8.3. Take out and put away the optical port blanks
SECTION: Clean Up
8.4. Take out the light
SECTION: Clean Up
8.5. Unscrew the bottom of the electrode chamber, careful not to let the electrode and magnetic follower fall
SECTION: Clean Up
8.6. Pull out the electrode disk, catch the magnetic follower
SECTION: Clean Up
8.7. Rinse the inside of the electrode chamber with dH2O
SECTION: Clean Up
8.8. Take the O-rings off the electrode and rinse them with dH2O
SECTION: Clean Up
8.9. Take off the membrane and paper from the electrode and dispose of them in the bio-waste bin
SECTION: Clean Up
8.10. Rinse the electrode disc with dH2O
SECTION: Clean Up
8.11. Rinse the magnetic flea with dH2O
SECTION: Clean Up
8.12. Make sure everything is gently and thoroughly dried, use a kimwipe for the electrode disc. 
SECTION: Clean Up
8.13. Repeat the same polish process as done at the beginning on the electrode/electrode disc. It's important to clean the electrode disc thoroughly right after you're done using it to prevent any tarnish buildup, which can happen after one day of use. The electrode disc should also be stored with a dehydration packet.
SECTION: Prepare 0.5M NaHCO3 Solution
1.2. Weigh out 0.420 g of sodium bicarbonate 
SECTION: Prepare the electrolyte
2.1. Weigh out 17.5 g of anhydrous KCl salt
SECTION: Clean Up
8.1. Remove the sample from the electrode chamber using a pasteur pipette