Isolation of Chloroplasts by Differential Centrifugation
Equipment and Supplies per team of two students:
clean sharp sand
50 mL 0.5 M sucrose (17% w/v)
cheese cloth, 12 x 12 inches
Styrofoam cooler for ice bath
25 mL graduated cylinder
mortar and pestle (or blender)
gtable top clinical centrifuge
glass filter funnel
two 16×150 mm test tubes in rack
three 13×100 mm test tubes in rack
plastic capped 15 mL centrifuge tube
double pan balance
glass stirring rods
KEEP ALL EQUIPMENT AND MATERIALS ICE COLD:
Per pairs of students:
Prepare, weigh and homogenize:
Grind 8 g deveined spinach with ½ tsp clean sharp sand in mortar and pestle to a paste.
Suspend in 0.5 M sucrose:
Measure out 16 mL ice-cold 0.5 M sucrose solution in a 25 mL graduated cylinder. Add in 3-4 mL increments, grind to smooth pulp with each addition. (A blender may be used for >100 mL volumes)
homogenate through about eight layers of clean cheese cloth in a glass funnel into an iced 16×150 mm test tube.
Pour filtrate back into 25 mL cylinder and record volume. Save ~0.5 mL of the filtrate (F1) in a labeled 13×100 mm test tube to examine at 400x under microscope to determine composition and illustrate in notebook. Note appearance of components and degree of heterogeneity. (Label cells, ghosts, chloroplasts, mitochrondria, debris.)
Centrifuge at low speed: prepare a balance tube against the filtrate in a 16×150 tube and spin at 50x g for 10 minutes (speed 2 on the clinical centrifuge).
Decant the top 10 mL into a clean cold centrifuge tube, discard sediment. Record volume. Save ~0.5 mL supernatant (S1) to examine under microscope to determine composition, illustrate and label as in step 2.
Centrifuge the supernatant from step 3 opposite a carefully balance tube at 1000x g for 10 minutes (speed 7) to precipitate chloroplasts. How does the supernatant appear? Precipitate? Carefully decant all of the supernatant into 16×150 mm tube but save the pellet. Discard supernatant if you have a significant pellet. (You will lose some soft pellet, but not to worry.)
Resuspend pellet from step 4 to 1/10th of the volume of the step 2 filtrate in ice-cold 0.5 M sucrose with a clean, ice cold stirring rod. Record final volume. Keep on ice at all times. Examine suspended organelles (SO) under microscope to determine composition, illustrate as is step 2. [You should have illustrations of F1, S1 and SO in your book.]