Chloroplast isolation

	ISOLATION OF CHLOROPLASTS BY DIFFERENTIAL CENTRIFUGATION ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
grind spinach with sand as a grinding aid	This page has been accessed times since 5 December 2002. 18 Nov 1992, 23 Nov 93, 22 Nov 94, 27 Nov 95, 20 Sept. '96, 26 Nov 96, 21 Nov 00, 20 Nov 01 From DBF Notebook, C.C. III, p. 84	decant after first centrifugation

Equipment and Supplies per team of two students:

SUPPLIES	EQUIPMENT
Fresh spinach clean sharp sand 50 mL 0.5 M sucrose (17% w/v) cheese cloth, 12 x 12 inches ice	Styrofoam cooler for ice bath 25 mL graduated cylinder mortar and pestle (or blender) gtable top clinical centrifuge glass filter funnel	two 16x150 mm test tubes in rack three 13x100 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)
	Filter homogenate through about eight layers of clean cheese cloth in a glass funnel into an iced 16x150 mm test tube.
	Pour filtrate back into 25 mL cylinder and record volume. Save ~0.5 mL of the filtrate (F1) in a labeled 13x100 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 16x150 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 16x150 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.]