© Dr. Bibit Traut
Botany 10 LAB
Spring 2011
Osmosis & Photosynthesis
I. Osmosis
1) Osmosis is the diffusion of water through a semi-permeable membrane. This process
is driven by the movement of water (the solvent) from a region where water is more
concentrated to region where it is less concentrated. In other words, water will move to
from regions of low solute concentration to regions of high solute concentration. The
process of osmosis is important in many plant processes, especially translocation and
transpiration.
2) Osmosis concentration experiment
a) Dialysis tubing will serve as the differentially-permeable membrane in this
simulation of artificial cells, allowing only the water to diffuse through it. You will
observe the effect of cells with differing solute concentration to illustrate the effect of
solute concentration on osmosis.
b) Procedure:
i) Cut three 12.5 cm pieces of dialysis tubing and soak them in DI water.
ii) Close one end of each dialysis tubing by tying it closed with string.
iii) Fill each tube with one of each of 3 different solutions
(1) 15 ml of sugar syrup solution (1:2 dilution)
(2) 15 ml of sugar syrup solution (1:4 dilution)
(3) 15 ml of distilled water
iv) Make sure dialysis tubing with its solution is not tight, but flaccid, then tie off the
other end to seal and rinse off with DI water (be sure string is wet too. Why?).
Dry bag and weigh to the nearest gram.
v) Place each bag in a separate 600 ml beaker with 400 ml of distilled water in it.
Make sure the beaker is labeled with the concentration of the solute in the
dialysis tubing.
vi) Weigh bags in grams (dried bag) initially and then at 10 minutes, 20 minutes,
and 30 minutes.
c) Does beaker water change color? Same for each bag? What had the greatest rate
of osmosis? How might plants use this osmotic gradient as a “pump”?
II. Guard cell response to osmotic pressure
1) The opening of guard cells is achieved by turgor pressure in these crescent shaped
cells. When water is readily available, the guard cells will take in water, causing them
to expand differentially due to cellulose fibers that run through them. When plants are
water stressed, a process initiates that changes the osmotic potential inside of the
guard cells and water leaves them, causing them to deflate and shut. You will observe
this process by simulating a change in water potential by adding a salt solution (5%
NaCl) to epidermal tissue.
2) Procedure:
a) Obtain a leaf specimen from your instructor (e.g. Zebrinia). Bend it in half with the
underside inward in order to cause the whole leaf to crack. Next use forceps to peal
off the thin epidermal tissue and mount on a slide in a drop of distilled water and
cover with a coverslip. Observe and sketch the stomata.
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© Dr. Bibit Traut
Botany 10 LAB
Spring 2011
b) What do you think will happen to the stomata if you add a salt solution to the
epidermis? Why? Add 5% NaCl to the specimen by drawing it through under the
coverslip (by applying a drop on one side of the coverslip and having a kimwipe on
the opposite edge of the coverslilp). Observe and sketch the stomata.
c) Questions/observations:
i) Why do you think stomata are open when water is plentiful?
ii) How do the stomata respond to changes in water potential/turgor pressur?
What mechanisms are involved?
iii) Why were you able to use a salt solution to mimic changes in water availablity in
a leaf that effects the stomata opening?
III. Photosynthesis
1) Relationship of Photosynthesis and CO2
a) Recall the relationship between photosynthesis and the utlilization of CO2. For this
experiment you will observe this relationship.
b) Procedure:
i) Fill a test tube with water and then add a dropperful of phenol red, so that the
solution turns pink/red. Phenol red is a pH indicator that turns red when pH is 7
or greater and yellow when pH is less than 7. Because of the
bicarbonate/carbonic reaction, when CO2 decreases the solution will be more
basic and the solution will be red, however when CO2 increases the solution
becomes more acidic and yellow.
ii) With a straw, blow into the tube until the solution turns yellow (acidic). What are
you adding to the solution by doing this?
iii) Empty half of this solution into another test tube and add a sprig of Elodea.
iv) Place both test tubes in bright light for ~20 minutes. How do the two tubes differ
after this time? Why did the color change in one tube and not in the other?
2) Paper Chromotagraphy
a) Paper chromatography is a technique that is used to separate plant pigments. This
process works by using the differences in charges of molecules and differences in
polarity of chromatography paper and non-polarity of petroleum ether solvent. The
pigments are more or less polar (see handout) and because of this can be
separated based on their differential attraction or lack of attraction to the
chromatography paper. Remember, that like attracts like (polar binds to polar,
nonpolar binds to nonpolar).
b) Procedure
i) You have pre-cut strip of chromatography paper. DO NOT TOUCH!! Use your
forceps to handle the paper. Fit the chromatography paper in your corked tube
so that it hangs about half a centimeter above the bottom. You will add solvent
to the bottom of the tube so that it will contact the paper, but be below a pigment
spot that you have made on the paper.
ii) Remove the paper and place pigment extract on the bottom of the paper using
the capillary tube to repeatably apply and make a dark spot (about 1cm up, so
that when the paper hangs in the tube, the paper will contact the small amount
of solvent in the bottom of the tube, but the pigment drop is above it).
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© Dr. Bibit Traut
Botany 10 LAB
Spring 2011
iii) Hang the paper in the tube and allow the solvent, being careful that when you
place the paper in, it does not go too deep and the pigment spot gets “washed”
out into the solvent. Next, the solvent will begin to move up the paper,
“grabbing” pigments as it goes and separating the 4 major photosynthetic
pigments. Remove the paper from the tube once the solvent reaches the
paperclip from which the paper is hanging.
c) Describe how the pigments separated and identify each pigment on your paper.
3) Relationship of Photosynthesis to Chlorophyll and Starch Accumulation
a) Photosynthetically active cells necessarily contain chlorophyll. The product of
photosynthesis accumulates and is stored as starch. Some plants are variegated
and have areas lacking pigment and therefore appear white or pink/red in parts and
green in other areas (with chlorophyll-containing cells). Coleus is an example of
such a plant. In this lab, you will utilize this difference to observe the relationship
between photosynthesis, chlorophyll and starch.
b) Procedure:
i) Obtain both a masked (covered for 5 days in foil) and unmasked Coleus leaf.
Sketch an outline of the leaf and note where the green parts are. You will repeat
the process below for each leaf and then compare the amount and distribution of
starch in each of them.
ii) Using tongs, immerse the leaf (either the masked or unmasked—KEEP TRACK)
in boiling water for ~10-15 seconds to break down the plasma membranes of the
cells. Then transfer the leaf to boiling ethanol to extract the chlorophyll from the
leaf. When the leaf has lost most of its pigments, remove it and rinse it with
water.
iii) Place the leaf in a Petri dish and add iodine dye so that the surface is covered
and the starch is stained. Describe the results, focusing on how the masked leaf
differed from the unmasked leaf and what causes this.
IV. Questions for Review
1. How does an osmotic pump work to move sugars throughout a plant?
2. Assume that the Elodea sprig in the phenol red solution carries on photosynthesis.
Does the oxygen given off have anything to do with the color change?
3. What serves as evidence that chlorophyll has been removed from the Coleus leaf?
4. During paper chromatography, what pigments were golden-yellow? What light would
be absorbed by these pigments?
5. How is osmosis involved in the opening of guard cells? How does water stress or
wilting of a plant affect the opening/closing of stomata?
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