laboratory
Activities:
INTRODUCTION
ACTIVITY 1. REVIEW OF PHOTOSYNTHESIS AND CHLOROPLASTS
ACTIVITY 2. THE ROLE OF PIGMENTS AND LIGHT
Background
  Chromatography
Pigments and Photosynthetic Activity
ACTIVITY 3. MEASURING THE RATE OF PHOTOSYNTHESIS
  The spectrophotometer
  Performing the experiment
  Data collection
ACTIVITY 4. THE PLANT LEAF: AN ORGAN DESIGNED FOR PHOTOSYNTHESIS

Performing the experiment

Our experiment will measure the rate of photosynthesis by isolated thylakoids under different lighting conditions. We will also examine the effect of boiling the thylakoids before use. For each light condition, a separate assay will be run in which absorption of the sample will be measured at 1-minute intervals for 10 minutes. A solution without thylakoids (for the 0 time point) and 5 experimental samples will be prepared. The table below lists the contents of the 6 tubes used in this experiment. View the next video to watch the tubes being filled.

Solutions/Light
Exposure
Test Tubes
No thylakoids

No light

White light

White light,
but boiled
thylakoids

Green light

Blue light

Phosphate buffer
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
Water
3 ml
3 ml
3 ml
3 ml
3 ml
3 ml
0.1% DPIP
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
Light conditions
white*
none
white*
white*
green
blue
Thylakoids
X
3 drops
3 drops
X
3 drops
3 drops
Boiled thylakoids
X
X
X
3 drops
X
X

* Sunlight or light from a laboratory lamp (includes all wavelengths within the visible spectrum).

video - filling the 6 test tubes

Thylakoids are added to each sample, contents of the tube are quickly transferred to a cuvette which is placed under a light source, and timing is begun. Absorption measurements are taken at 1-minute intervals for 10 minutes. In the case of the "no light" sample, the cuvette is immediately covered with aluminum foil. It is kept in constant darkness, except for the few seconds that the cuvette is uncovered and placed in the spectrophotometer. In one sample, the thylakoids are boiled before use.

white

During the timed period, each sample is placed near a lamp (a white light source). Colored plastic sheets are placed in front of the lamp to provide green or blue light. A water bowl is placed between the sample and lamp to protect the sample from heat; the lamp is hot, but most heat is absorbed by the water.

The arrow points to the cuvette containing an experimental sample.

White light treatment
green light
blue
Green light treatment
Blue light treatment

View the following video the watch the start of an assay.

video - starting an assay

Data Collection

The spectrophotometer has been used to obtain measurements from the "no light", white light,green light, and blue light treatments. The spectrophotometer wavelength was set at
610 nm. and absorption was recorded for 10 minutes in each case.

Your technician has recorded all absorption values and placed them in a table on your worksheet.

Use the on-line plotter to graph the data as follows:

Graph l - Plot time vs. absorption for no light, white light, and boiled thylakoids. You will need three Y columns. Use lines to connect the points so that the three lines can be compared.

Graph 2 - Plot time vs. absorption for white light, green light, and blue light. You will need three Y columns. Use lines to connect the points so that the three lines can be compared.

Submit these graphs for questions 11 and12.
Label all Y-column headings to receive full credit (the plotter will make a legend for you).

Carefully examine your graphs, then answer question 13.

Examine the photograph below, then answer question14.

cuvettes This photograph of cuvettes was taken at the end of the photosynthesis experiment.
Cuvettes from the photosynthesis experiment