TITLE: Algaeorithm Classroom Guide: Constructing Mini Photobioreactors for Microalgae
AUTHORS: Ashwin Mukherjee, rohan chanani, Jacob J. Valenzuela

[DESCRIPTION]
A guide to constructing mini photobioreactors and cultivating algae in classroom settings.

[STEPS]
SECTION: Prepare Photobioreactors
1. Place the glass tube rack and unplugged air pump on a table or flat surface where they will not be disturbed. 
SECTION: Sample Photobioreactors + Prepare Hemocytometer
3.  Combine 3 parts isopropyl alcohol and 7 parts water in a spray bottle
SECTION: Analyze Samples
10. Place the 10x eyepieces into the binocular ports of the microscope. 
SECTION: Prepare Photobioreactors
2. Repeat the following procedure for each reactor you plan to prepare:
SECTION: Prepare Photobioreactors
2.1. Remove the cork from one glass tube and set the tube aside.
SECTION: Prepare Photobioreactors
0.29. Drill two holes in the cork using a drill bit. The first should be at least   in diameter, and the second should be equal or lesser in size. Make sure that the holes are separate from each other and separate from the edge of the cork. 
SECTION: Prepare Photobioreactors
2.2. Thread a section of aquarium tubing through the larger opening in the cork. Do not cut the tubing until you are sure it will be long enough to connect the pump to the reactor. The amount of tubing exiting the cork on the bottom should be less than an inch from the bottom of the tube when the cork is placed back onto the tube. 
SECTION: Prepare Photobioreactors
2.3. Remove the cork with tubing from the test tube and set it aside.
SECTION: Prepare Photobioreactors
2.4. Using a dropping pipette (like the one provided in the hemocytometer kit), pipette approximately 35 mL of the growth media (concentrated or freshwater) and approximately 6 mL of the Chlamydomonas culture into the glass tube.
SECTION: Prepare Photobioreactors
2.5. Place the cork with tubing back onto the glass tube so that the tubing is within an inch of the glass tube’s bottom and place the entire apparatus onto the glass tube rack.
SECTION: Prepare Photobioreactors
2.6. Connect the tubing to the pump using one of the following methods
If using gang valves, connect the open end of the tubing to one of the gang valves and use another section of tubing to connect the gang valves to the air pump. 
If not using gang valves, directly connect the open end of the tubing to the aquarium pump output. 
SECTION: Prepare Photobioreactors
2.7. Plug in the aquarium pump and adjust the aquarium pump output until the reactor is bubbling gently.

 

SECTION: Sample Photobioreactors + Prepare Hemocytometer
4. Clean the glass section of the hemocytometer and 1 hemocytometer coverslip using the sheets provided with the hemocytometer.
SECTION: Sample Photobioreactors + Prepare Hemocytometer
5. Place the coverslip onto the counting section of the hemocytometer.
SECTION: Sample Photobioreactors + Prepare Hemocytometer
6. Unplug the aquarium pump.
SECTION: Sample Photobioreactors + Prepare Hemocytometer
7. Carefully remove the top from one photobioreactor and use a pipette to pick up approximately10 µL or 1 drop.
SECTION: Sample Photobioreactors + Prepare Hemocytometer
8. Pipette approximately 10 µL into one side of the hemocytometer. The drop should disperse evenly under the coverslip.

 

 
SECTION: Sample Photobioreactors + Prepare Hemocytometer
9. Set the hemocytometer aside and replace the top of the photobioreactor.
SECTION: Analyze Samples
11. Rotate the objective lenses until the 10x objective lens is directed at the observation area of the microscope.
SECTION: Analyze Samples
12. Use the coarse focus knob to lower the observation area as much as possible.
SECTION: Analyze Samples
13. Securely place the hemocytometer in the observation area of the microscope. Turn the microscope on and adjust the light to the dimmest setting. 
SECTION: Analyze Samples
14. Adjust the binocular eyepieces until you see one image while looking through them. 
SECTION: Analyze Samples
15. While looking through the binocular eyepieces, slowly raise the observation area using the coarse focus knob until an image forms. Once an image forms, use the fine focus knob to clarify the image.  
SECTION: Analyze Samples
16. Rotate the objective lens to 40x, ensuring that the objective and the slide DO NOT TOUCH. Use the fine focus knob to refocus the image.

 

 
SECTION: Analyze Samples
17. Collect data
SECTION: Analyze Samples
17.1. If collecting manually, follow the instructions included in the hemocytometer kit to calculate cell concentration.
SECTION: Analyze Samples
17.2. If collecting automatically through Algaeorithm, follow the linked guide to capture/upload images and extract cell data.