ISOLATION OF CHLOROPLASTS BY DIFFERENTIAL CENTRIFUGATION ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry              University of Cincinnati Clermont College,  Batavia OH 45103

Equipment and Supplies per team of two students:

Fresh spinach
mortar and pestle (or blender)
clean sharp sand
50 mL 0.5 M sucrose (17% w/v)
Styrofoam ice bath
25 mL graduated cylinder
cheese cloth, 12 x 12 inches
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
table top clinical centrifuge
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.]

Copy this table into your notebook prior to starting the chloroplast reduction experiment. Every blank square gets a reading.
 

vol. chloroplasts

light cond

Time of exposure in minutes

0

1

2

3

4

5

6

7

8

9

10

15

0

dark

x

x

x

x

x

x

x

x

x

x

0

light

x

x

x

x

x

x

x

x

10uL

dark

x

x

x

x

x

x

x

x

x

x

10uL

light

50uL

dark

x

x

x

x

x

x

x

x

x

x

50 uL

light

See previous protocol for the preparation of the purified chloroplasts. Continue to keep them ice cold until the moment you add them to the prepared tubes. Teams of two.
 

Supplies:

purified spinach chloroplasts, ice cold
0.1 M PO₄ buffer, pH 6.5 (pH 7.0 OK?)
2.5 x 10^-4 M 2,6 dichlorophenolindophenol
(36.3 mg DCIP/500 mL, A₆₀₀ 3.0)
displacement pipets
six unblemished 13x100 mm test tubes
100 watt light 1/ two teams
cuvette for water blank 1/team
37C incubator (can exclude light)

Set up hot block next to spectrophotometer:
hot block, 37C 1/team
spectrophotometer 1/team

1. Set up apparatus:
a: 37C hot block (for 13x100 mm tubes) nearby and warmed up.
b: 37C incubator warmed up.
c: spectrophotometer on the same desk as the light exposure apparatus.
d: 100 watt bulb with reflector 25 cm from open test tube rack.

• Construct a data table in your notebook to accept data (previous page in handouts).

• Prepare DCIP reaction mix which should have an A₆₀₀ of 0.400 to 0.600.

4. Dispense 4 mL of the reaction mix to each of your tubes with repeater pipetter.
5. Add all of the following ingredients except for the chloroplasts:
 

6. Prewarm prepared tubes (lacking chloroplasts) in 37C hot block for 2 min.
7. Add diluted chloroplasts to tubes 3 and 5. Mix and read the A₆₀₀ of 1, 3, and 5 against a water blank (= T₀ for dark tubes). Place in 37C incubator, keep light excluded.
8. Add indicated volume of chloroplasts to tubes 2, 4 and 6. Mix and read A₆₀₀ of these tubes as in step 4 ( = T₀). Place 25 cm from a 100 watt bulb (set up next to the spectrophotometer).

9. Read A₆₀₀ of 4 and 6 every minute for 10 minutes, read tube 2 only at 0, 5, and 10 min. (Keep tubes clean, read in consistent configuration.)
10. Read A₆₀₀ of tubes which were kept in the dark again at 15 minutes.
11. Plot the absorbency of each tube versus time.

• Discuss the significance of the differences between the various six curves.

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