Photosynthes lab ( introduction + objectives + methods )

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GES 102 – PHOTOSYNTHESIS LAB

This week you will investigate the effect of light intensity on the rate of photosynthesis in
spinach leaves. Fresh spinach bought in the store is green, and the leaves still have the
ability to do photosynthesis. Photosynthesis produces carbohydrates and oxygen and we
will use the rate of oxygen production as an indicator of how quickly the spinach leaves
actually do photosynthesis.

You will use a hole punch to make a number of small disks of spinach. These disks float
when they contain oxygen from photosynthesis. By applying vacuum to the small spinach
disks, the oxygen is forced out, and they sink. When you then place the disks in a
transparent cup with water, and expose them to light, they will produce oxygen and
eventually float. The time it takes the small disks to float is a measure of photosynthesis,
and you will test the effect of different light intensities on the floatation time.

Background:

In your first lab this semester you investigated how the UV portion of sunlight was able
to cause harm to living cells, which is not so surprising since this shorter wavelength
carry enough energy to damage the cell’s DNA. In this week’s lab we will instead use the
portion of radiation from the sun that we call daylight (380-780 nm), plenty of which
makes it down to the earth’s surface. Our eyes are sensitive to this portion of the
spectrum, and it’s also the portion plants use for photosynthesis.

The process of photosynthesis can be simplified as:

CO2 + H2O  sugar + O2.

It is really divided up into two sets of reactions, the Light Reaction and the Calvin
Cycle. In the Light Reaction the plant cells use chlorophyll to capture some of the energy
in sunlight. When sunlight hits a chlorophyll molecule, electrons in the chlorophyll

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molecule are excited, they ‘spin’ faster because of the absorbed energy. These excited
electrons are passed on down the line and chlorophyll will take a continuous supply of
new electrons from H2O which is broken down, producing O2. The energy in the excited
electrons is eventually used to produce two high energy molecules, called ATP and
NADPH. In the next step, the Calvin Cycle, the energy contained in ATP and NADPH is
used to help take CO2 (carbon dioxide) from the air and manufacture sugar.

Spinach plants normally get their CO2 from the air, through small openings on the
underside of their leaves, called stomata. In our experiment some sodium bicarbonate
(NaHCO3) will be added to the water, and provide the carbon needed for the small
spinach disks to do photosynthesis. The O2 produced will be trapped inside the spinach
disks and increase their buoyancy until they float.

Alongside photosynthesis, the plants also do cellular respiration. They certainly are
autotrophs and can produce their own “food”, the sugar. But they also need to break
down the sugar in order to get the energy required to pay their energy bills, i.e. they need
also to be heterotrophs, just like animals. It should be no surprise to find that plants cells
have both chloroplasts and mitochondria. In our experiment with the spinach disks, some
cellular respiration will happen along with the photosynthesis. This will make it take a bit
longer for the disks to float, and our measurement of photosynthesis is really a measure
of net photosynthesis.

You will also place some of your floating spinach disks in the dark. Photosynthesis is
obviously impossible in the dark, and now the spinach disks can only do cellular
respiration, which can be simplified as:

sugar + O2 CO2 + H2O.

In the dark the spinach disk will consume O2, and once they have consumed enough O2,
their buoyancy will have decreased enough for the spinach disks to sink. You will
determine how long time this takes. Gross photosynthesis is the total photosynthesis
undertaken by the spinach disks, where you add the net photosynthesis + cellular
respiration (as measured in the dark).

Materials:

• 0.2% sodium bicarbonate solution (NaHCO3), with a few drops of dishwashing
fluid to lower the surface tension of the water (and allow it to enter the spinach
disks)

• 4 clear plastic cups
• Quantity of fresh spinach leaves
• 10 cc plastic syringe
• Timer
• 4 clear plastic cups
• Hole punch
• Light banks with three different light intensities

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Experiment:

Preparing the spinach disks for each experiment:

1. Take a spinach leaf and then use a hole punch to punch out at least 10 small
spinach disks from the leaf. Make sure to avoid any major veins in the leaf.

2. Now remove the plunger from the syringe and carefully place the 10 spinach

disks inside the barrel.

3. Slowly push the plunger back into the syringe, which will make the plunger
slowly push the spinach disks into the syringe. Take great care not to crush the
disks, and continue until there is only a small amount of air left with the disks at
the end of the barrel.

4. Now put the tip of the syringe into the sodium bicarbonate solution and pull a

small amount of fluid into the syringe. Tap the syringe and check that the spinach
disks free from the walls of the syringe and intact. They should be floating on top
of the fluid.

5. You are now ready to apply a vacuum to the disks. Hold a finger over the syringe

opening and at the same time very slowly draw back the plunger. This creates a
vacuum in the space with the spinach disks. Hold this vacuum for about 10
seconds.

6. The vacuum should draw some sodium carbonate solution into the leaf disks,

replace the O2, and cause the leaf disks to sink. You may have to repeat the
vacuum steps 2-3 times to make all the disks sink.

Starting the experiments:

1. There are three light banks in the lab, each with a different light intensity, and it
doesn’t matter in what order you use them. You need to perform one floatation
experiment with 10 spinach leaf disks using each light bank, and measure how
long it takes for all the leaf disks to float in each light intensity. What is your
hypothesis regarding the results?

2. You also need to do one experiment where you measure cellular respiration in the

leaf disks, by putting them in the dark and measuring how long it takes for the
disks to sink. This takes longer than making the disks float, and you should start
this particular experiment early in the lab period.

3. A good strategy is doing the cellular respiration experiment with the disks you

made to float using your first light bank. It doesn’t matter what particular light

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intensity you happened to do first. After making sure all leaf disks are floating,
put them in the dark, and briefly look at them after each minute has passed, noting
how many have sunk, and continue until all leaf disks have sunk to the bottom of
the cup.

4. For each of the three light intensities, record the number of disks that are floating
at the end of each minute, and make sure to swirl the disks to dislodge any that
may be stuck against the sides of the cups. Continue up to a total of 15 minutes.

5. An example of a data table is found below, but make sure to enter your results
into your lab notebook. For the spinach disks you put in the dark, you need to
continue recording until a total of 30 minutes, unless all the leaf disks have sunk.

6. You also need to make a graph of your results from each of the three trials, and
make sure to analyze the results.

Minutes # of disks floating

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

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You need to answer the following questions in the lab notebook. As before, don’t copy
the question as such, instead write a complete sentence that includes the information
asked for.

1) What was the function of the sodium bicarbonate in this experiment?

2) What process causes the leaf disks to rise?

3) Which trial worked the best?

4) What was the purpose of putting some leaf disks into the dark?

5) How does light intensity affect the rate of photosynthesis?

References

Adapted from Biology: Exploring Life. Teaching Resources, by Brad Williamson
(http://www.elbiology.com/labtools/Leafdisk.html).

© John Hakanson (2014)