Materials

  • Fresh Spinach
  • Cheese cloth or coffee filter
  • 10x 2 mL centrifuge tubes
  • Pasteur transfer pipettes
  • Beaker
  • Ice
  • 100 mL 0.5 M sucrose (17% w/v), store on Ice
  • 80% acetone

Equipment

  • Chopping board and knife, scissors will also work
  • Mortar or blender
  • Centrifuge
  • Microscope or spectrophotometer

Procedure

  1. Wash 10 grams of spinach leaves
  2. Cut out the veins of the leaves
  3. Grind the leaves in a mortar or blender
  4. Add 5 mL of 0.5 M sucrose to the solution and mix
  5. Filter the mixture through the cheese cloth or coffee filter into a beaker
  6. Save 0.5 mL of the filtrate in a tube for comparison later on
  7. Transfer two times 2 mL to two centrifuge tubes, make sure they’re balanced
  8. Centrifuge at low speed (50x - 200x g) for 3 - 10 minutes to get rid of cell debris (white pellet)
  9. Transfer the supernatant to two fresh centrifuge tubes, save 0.5 mL of each tube for later examination
  10. Centrifuge at high speed (1000x g) for 10 minutes to pellet the chloroplasts
  11. Dispose the supernatant when you feel satisfied with the pellet, spin longer if you feel like it
  12. Resuspend the pellet in 0.5 mL 80% acetone
  13. Examine the samples under the microscope and/or measure the absorbance at OD650. You can count the number of Chloroplasts per mL

Return to BHA6 - class 7