Download Teacher Instructions - 4UBiologyCourseWork

Teacher Instructions
0.1M Phosphate buffer: 174 g K2HP04 (dibasic) brought to 1 liter with
distilled H20 and 136 g KH2P04 (monobasic) brought to 1 liter with
distilled H20 (monobasic has a lower pH). Mix some monobasic with
dibasic until the pH is 6.5 (try 685 mL of monobasic into 315 mL of
dibasic). Since this solution is 1 M, 0.1 liter of the solution must be
diluted with O.9 liter distilled H20 to prepare a 0.1 M solution.
Sucrose: Add 85.5 grams of sucrose to 500 ml of distilled water. Store
in refrigerator.
DPIP (2,6-dichlorophenol-indophenol): Add 0.036 grams of DPIP to
500 ml of distilled water. Store in the refrigerator in an amber bottle or
a bottle wrapped in foil.
Buffer: Make a solution of 87 grams K2HPO4 (dibasic) brought to 500
ml with distilled water. Make a second solution of 68 grams KH2PO4
(monobasic) brought to 500 ml with distilled water. To make a stock
1.0 M buffer, mix 345 ml of the monobasic solution with 160 ml of
dibasic. Adjust the pH to 6.5. If the pH is too high, add more
monobasic. If the pH is too low, add more dibasic. In this lab,
you will need 0.1 M buffer, so take 10 ml of the 1.0 M stock buffer and
add 90 ml of distilled water. The stock keeps well at room
temperature.
Chloroplast suspensions: To prepare and prime the chloroplasts,
incubate fresh spinach leaves under a light for a few hours. Do not
allow the leaves to become hot. Pour 0.5-M cold sucrose into a
blender so that it just covers the blender blades. This is probably 100
mL or 200 mL of solution. Pack fresh spinach leaves into the blender
to a level one inch above the blades. Set up a beaker in ice with 2
layers of cheesecloth folded over a funnel. Blend spinach (about
three short bursts - errrrrrr; errrrrr; errrrrrr!). Squeeze through
cheesecloth into a large funnel leading into a large beaker that is
placed in an ice bucket.
Test the chloroplast suspension prior to use. If the DPIP is reduced too
rapidly, further dilute the suspension before distributing it into the
vials.
Preparation time required
Prior to class, allow about one hour to mix the buffer solution and
make the chloroplast suspension and pigment solution. The best
results are obtained when these materials are freshly made, but they
may be prepared the day before, and then be refrigerated until
needed for the lab. To get the best results from the chloroplasts,
incubate spinach leaves under a light for a few hours before use.
(Do not allow them to become hot.) The solvent used in part 2 will
evaporate unless kept in a sealed container. The buffer lasts
indefinitely and may be made well in advance. Additional time will
be needed to set out materials and clean up afterwards.
Be sure to turn the spectrophotometers on before the beginning of
class, b. They will need to warm up for at least 20 minutes.
note that above 600 nm, you have to shift the lever at the lower left
of the machine).
100-watt floodlight with large beaker of water in front for a heat
shield.
EXTENSIONS
1. Use colored filters around the cuvettes to test the effect of red,
blue, and green light on the
photosynthetic activity of spinach.
2. Vary the distance of the floodlight source to determine the effect
of light intensity on photosynthesis.
3. Compare the photosynthetic activity of spinach with that of
chloroplasts from other plants.
4. How does temperature affect the photosynthetic activity of
spinach?