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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, |
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.
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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 treatment |
Blue light treatment |
View the following video the watch the start of 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.
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This photograph of cuvettes was taken at the end of the photosynthesis experiment. |
Cuvettes from the photosynthesis experiment |