* Turn on the spectrometer at least twenty minutes before you need it.

1. Before you begin to use the spectrometer, you could concentrate the culture if you have a centrifuge. This gives you a better reading in the spectrophotometer. If you do not have one disregard steps a-c.

a. Place 50 ml of each of the three cultures in to three different centrifuge tubes. ( you need to put 50ml distilled water in another centrifuge tube for balance)
b. Place all of the tubes in the centrifuge and close the cover tightly.
c. Set the time for ten minutes.

1. Mark three cuvettes as sample 1, 2, and 3

2. Remove 45ml from the culture in the centrifuge tubes, (Precaution - If you concentrated the culture, do not shake it and mix it before this step) leaving 5ml of concentrated culture to be placed in one of the cuvettes.

3. Place the sample 1 culture in the cuvette 1 up to the line on the test tube, and follow this procedure for the for the other two samples as well..

4. Place same amount of water into a fourth cuvette.

5. Be sure you wipe the outside of each cuvette before placing them in the spectrophotometer. Use a pipette to mix up this solution in each cuvette.

6. You need to begin by setting your dial on the spectrophotometer for 664nm. Then you place the cuvette with water in the holding place.  Adjust the knob to zero the absorbency level.

7. Remove the water cuvette and place sample one in the holding place. Record the absorbency reading.

8. Reset the dial for 647nm. Again place the water cuvette in the spectrophotometer and zero the absorbency level.

9. Place sample one cuvette back in the holding place and record the absorbency level for 647nm.

10. Repeat these directions for samples 2 and 3.

11. When you finish recording all the findings, use the following formula to determine the chlorophyll a levels.

Green algae: 11.93A664 - 1.93A647 (multiply the absorbency level for 664 times 11.93 and subtract the product of 1.93 and absorbency for 647)

12. To find the chlorophyll b levels use this formula:

Green algae: 20.36A647 - 5.54A664 (multiply the absorbency level for 647 times 20.36 and subtract the product of 5.54 and absorbency for 664)

13. Multiply the answers for each chlorophyll a finding by 10³ if you began with a concentrated mixture. If you began with an unconcentrated mixture you multiply by 10⁴. Record your findings on the chart included.

14. If you wish to determine the ratio of chlorophyll a to chlorophyll b, you need to divide the answer for b into the answer for a. Then you multiply that answer by 10^-1 .

Using low power on the microscope, view the lined grids present on the chamber. There is an identical grid on either side of the moat. Examining the grid on one side of the moat. Notice there are nine large squares, each 1 mm on a side enclosed in the grid. IMPORTANT: You will be working with four squares only - those found at the four corners of the chamber.

Each of the corner squares is divided into 16 smaller boxes. Count all the cells in these 16 boxes on EACH corner.