Materials

• Chloroplast suspension from Exercise 8.1
  
• Acetone
  
• Spectrophotometer and tubes
  
• Clinical centrifuge and tubes

Procedure

1. Pipet 1.0 ml of a chloroplast suspension into a 15 ml centrifuge tube and add 8.0 ml of acetone. Mix thoroughly.

2. Add 1.0 ml of distilled water, mix again and centrifuge at 1000 xg for 3-5 minutes.

3. Turn on a spectrophotometer, adjust the wavelength to 652 nm and blank the instrument with water.

4. Measure the absorbance of the supernatant from step 2.

5. Calculate the amount of total chlorophyll in 1.0 ml of the chloroplast suspension. 2 The amount of chlorophyll is givenby the equation:

   mg chlorophyll		Absorbance
   ----------------	=	----------------
   ml		34.5

6. Calculate the dilution factor for your original chloroplast suspension so that when diluted with suspension solution, the final concentration of chlorophyll will be 0.01 mg/ml.

   All subsequent work with chloroplasts will use the diluted suspension so that the criterion of step 6 is met.

7. Use a hemacytometer to determine the number of chloroplasts per ml of any diluted suspensions.