A Sample Abstract from the Fall 1996 class is available.
In this exercise, you may feel insulted by the simplicity of the instructions and the simplicity of the exercises, but some of us need a review at the start of the semester to refocus our science skills in general. Many of us may also need to be reminded of the special differences between plant cells and the cells of other organisms. The plant cell is the unit of plant life and therefore a unit of plant function which means plant physiology! So this is the appropriate place to start a course in plant physiology.
1. Plug in
2. Turn on lamp, adjust brightness
3. Use coarse focus to lower the stage
4. Rotate 10x objective into position
5. Put slide on stage in clips
6. Use stage controls to center specimen in circle of light
7. Position condenser 1 mm below slide
8. Use coarse focus to position slide as close to objective as possible
9. Looking through ocular, use coarse focus to drop stage and bring specimen into focus
10. Use fine focus to focus sharply and to "optically" section specimen
11. If needed, close diaphragm partially to improve resolution
12. Rotate to 40X objective
13. Focus ONLY with fine focus
14. Increase light with knob if needed
15. If more magnification is needed:Rotate objectives between 40x and 100x16. When finished:
Place drop of oil in center of circle of light
Rotate 100x objective into position
Use FINE FOCUS ONLY !
Clean up afterwards with lens tissueRotate to 10x objective
Lower stage with coarse focus
Remove slide
Clean up microscope as needed
Cover microscope
Return microscope to storage area
Observation:
Following the instructor's directions, make a freehand section of a Forsythia stem. Prepare your own wet mount and observe. This means make a quick sketch of the overall tissue showing regions with obviously different cell types.Question:The color of the freshly mounted wood is: ________________
Can the color of cell boundaries be altered with a dye?Hypothesis:
The color of cell boundaries can not be altered with a dye.Prediction:
If the color of cell boundaries can not be altered with a dye, then the color of the section on the microscope slide should not be altered by remounting it in Phloroglucinol HCl.Experiment:
Remove the coverslip from your wet mount. Blot the stem section dry with a bit of paper towel. Add one drop of 5%(EtOH) Phloroglucinol, let stand 30 seconds, add one drop of 9N HCl, replace the coverslip and observe over the next 10 to 15 minutes; make a sketch.Analysis:Is this a true experiment? Yes No If no, what is lacking?_____________________
Our approach is strong in that we have no variation: our control is the very same piece of tissue as our treatment tissue. How could we further strengthen the approach? (Hint: is the treatment manipulation strictly parallel to a untreated control?)
______________________________________________________________________
______________________________________________________________________
The color of the wood cell boundaries treated with Phloroglucinol HCl is ____________
Decision:
The color of the treated wood cells is the same as
different thanthe untreated wood cells.
The hypothesis: "The color of cell boundaries can not be altered with a dye"Lignin has been shown to be a component of cell walls, especially secondary walls of wood cells, and along with cellulose these two components make wood very strong. The cell walls of wood cells provide the strength in the wood of a chair to hold your weight up against the force of gravity. One last observation might help convince you that the cell walls provide support: in any of your microscope observations did you observe materials inside the wood cell walls? Examine a prepared slide of Pinus (white pine) wood cells. Wood cells lack any other cellular component because these cells are dead, thus all of the strength of the wood is due to the wall alone. Wood cells are an example of sclerenchyma cells.is: rejected not rejected
You now have some experience with artificial colors in the microscope and you also have done a chemical test. It turns out that Phloroglucinol causes a color reaction in chemicals which have a particular chemical structure. One chemical which has the correct structure to cause a reaction is lignin. We could test the hypothesis that wood contains lignin. Treat a toothpick with phloroglucinol. What happens?
Would a color test be sufficient to conclude that wood contains lignin? yes no
If no, why not?___________________________________________________________
______________________________________________________________________
Please note: not all cells in a plant are alive at physiological maturity!! The conducting cells in wood, called xylem vessels or tracheids, are dead at physiological maturity. When they were alive they did not conduct water and minerals from the soil to the leaves. Note that not all cells in the Forsythia stem possessed large amounts of lignin! Cells are not all the same even in the walls!
Another part of the boundary is a cell membrane which textbooks tell you is made of phospholipid bilayers. Since plant cells have a cell wall, the membrane is not visible unless we pull it away from the wall slightly. Conveniently, the rigid cell wall therefore serves as a marker to show us when we have effectively altered the size of the cell membrane.
Observation:
Make a wet mount of an Mnium (moss) leaf and observe (sketch) the form of leaf cells in distilled water. Save the mount for later comparison.Question:
Are these cells dead or alive?Hypothesis:
These cells are alive.Prediction:
If these cells are alive, they should have a functional cell membrane which can exclude certain chemicals or ions (such as sodium and chlorine ions) but not others (such as water).Experiment :If the prediction holds:
What should happen to cells in a hypertonic solution? shrink enlarge
What should happen to cells in a hypotonic solution? shrink enlargeIf our prediction is completely backwards (the cells exclude water but not ions):
What should happen to cells in a hypertonic solution? shrink enlarge
What should happen to cells in a hypotonic solution? shrink enlarge
Make another wet mount of Mnium leaf but this time using 6% sodium chloride. After observing for two minutes, compare the cells in the salt solution with those in distilled water by recording the relative sizes and position of the membranes. Make a conclusion about whether the cell membrane was permeable to water or permeable to ions.Analysis:
Treatment Membrane Size Membrane Position Relative to Cell Wall 6% NaCl larger
smallerthan in 0% NaCl up against
separated fromthe wall 0% NaCl larger
smallerthan in 6% NaCl up against
separated fromthe wall
Decision:
The ions were excluded more
the same
lessthan water from the cells.
The hypothesis: "These cells are alive is: rejected not rejectedWhy did we not observe enlargement or bursting of the cells in distilled water?
We have observed cell membranes and cell walls in plants. One boundary layer is rigid and static, the other flexible and dynamic. A cell is more than its boundaries; it also must have something inside...
Examine the interior of the cells of a freshMnium leaf. There are several obvious internal structures (organelles).Question:We could take these textbook observations as facts, but as scientists we would be more convinced after testing their validity. To prepare ourselves for this test we need one more observation.
What color are these obvious structures?
What chemical is most likely observed?
What chemical process is carried out in these organelles?
What is the organic product of this chemical process?
What is the macromolecule polymer of this product?
On another microscope slide place a tiny amount of corn starch, add a drop of water and observe the starch grains. Apply a drop of iodine (I2KI = 5% I2 and 10% KI) stain to the edge of the coverslip and draw it under the coverslip.With these observations in mind, use the scientific method to verify the function of the obvious internal structures of a Mnium leaf.What color does iodine produce in combination with starch?__________________
_______________________________________________________________________Hypothesis:
_______________________________________________________________________Prediction:
If the hypothesis is true, then________________________________________________Experiment:when___________________________________________________________________
Control:_________________________________________________________________Analysis:Treatment:_______________________________________________________________
Results:
Control:__________________________________________________________Treatment:________________________________________________________
[if you did not get what you expected...why not?]
_______________________________________________________________________Decision:
The hypothesis above is: rejected not rejected
Most mature plant cells have a distinct cell wall, some thin and others thick. Cells of leaves have chloroplasts, but do all plant cells have chloroplasts? Your instructor will demonstrate how to make a peel of a leaf epidermis from a plant called Rhoeo discolor. The upper epidermis of the leaf is essentially transparent but the lower epidermis is quite purple. Make a wet mount of the upper and lower epidermal layers peeled from this leaf and observe (sketch) for comparison. The kidney-shaped cells common in the purple lower epidermis peels are called guard cells. The other cells of the epidermis are simply common epidermis cells. Try to answer the following questions through observation and/or experimentation:
Are there any natural openings through the epidermal layer you have peeled off?Together, the cell wall, the chloroplasts, and the large vacuole you have observed are three very distinct cell structures lacking in most animals and many fungi. Most of the other cell structures (nucleus, mitochondria, endoplasmic reticulum, ribosomes, Golgi bodies, etc.) are common to all eukaryotic cells including plant cells.
Do any openings occur in a particular location?
Compare the contents of the common epidermis cells and the guard cells.
Can you tell whether the purple pigment is contained in a particular cellular compartment?
What treatment would help observe membranes more easily? (do it!)
Do you think the vacuole is well-named?
Can you observe the cell nucleus in either cell type? (adjust the iris diaphragm!)
How many nuclei are there per cell among those having visible nuclei?
You have now observed some of the major organelles of a cell, but you might not have observed mitochondria. It seemed easy to locate cell walls using a stain for lignin, to find the chloroplast by staining for starch. The mitochondrion is more difficult to observe: it is smaller than nuclei or chloroplasts and lacks colorful organic chemicals. It is, of course, the powerhouse of the cell and therefore must have enzymes which release the energy stored in organic molecules. Perhaps we could stain for the special enzymes of the energy pathway. This might add to our theory about their living state.
Observations:
0.15%(aq) methylene blue is known to change color in the presence of electrons and hydrogen ions. The respiration pathway includes enzymes such as succinate dehydrogenase which liberate electrons and hydrogen ions. Thus methylene blue might reveal mitochondria to our microscopic probe.Question:
Do plant cells contain mitochondria?Hypothesis:
Plant cells lack mitochondria.Prediction:
If plant cells lack mitochondria, then applied methylene blue should not stain the mitochondria and thereby reveal their location.Experiment:
Mount a fresh upper-epidermis peel of Rhoeo in 0.5% methylene blue and observe (sketch) immediately and over the next several minutes. Look closely in clear areas between any chloroplasts...mitochondria are SMALL!Analysis:
Don't forget to make some mental comparisons between control and treatment!Decision:
The hypothesis: "Plant cells lack mitochondria" is: rejected not rejected
To answer this question a good scientist selects an organism that can help us test the idea. Physarum is a slime mold (Kingdom Protista) that has very large cells (the better to see your cytoplasmic streaming my dear). In fact the cell is large enough to fill a Petri dish (and more!).
In the laboratory are incubation solutions that include a high calcium concentration (10
For our action potential we need to put some electrical current across the cell. The cell will be connected to wires leading to a 1.5 V (AA) battery. In an attempt to force the electricity through (rather than around) the cell, we need to touch the surface of the cell directly with one of the wires. Be careful not to penetrate the cell and go into the agar medium beneath. The other wire will rest embedded in the agar medium. When the first wire touches the cell an ion-based circuit will be closed; the circuit goes through the medium, the cell, and then to the touching wire. A brief contact with the cell would constitute an action potential across the cell.
Work with your partner to observe the effects of applying electrical current across a cell that is showing active cyclosis (cytoplasmic streaming). The observer needs to locate an area of active cyclosis through the microscope. The observer continues to observe while giving a signal to the partner to touch the wire briefly to the cell. Observe for an additional minute or so at 15 sec intervals. After cyclosis resumes, the partners should change roles and repeat the observations.
You might then compare observations with some additional calcium available to the local region of the cell. You might also try some EGTA (a calcium chelating agent).
Congratulations, you made it through some cytochemistry of dead cells through static chemical detection in living cells, to ion-induced plasmolysis, to an enzyme-linked staining system, and finally the role of ions and electrical potential in movement inside a living plant cell. We moved from simple chemistry to plant physiology!
Meristematic:
Go back to the Course Schedule.
How do you think the calcium and electrical potential are functioning in cyclosis in this project? Does it seem backwards? Why would this be so?
Observations of cyclosis in the presence of 10-4 M Ca2+:
Observations of cyclosis in the presence of 10-7 M Ca2+:
Observations of cyclosis in the presence of EGTA:
More on Plant Cells
cells specialized in mitosis functions,
living at maturity, rapid mitosis (short G1, S, G2 interphases), extremely thin cell wall, no vacuole, usually isodiametric in shape but very small in diameter
Parenchyma:
cells specialized in a variety of ways (the typical cell?),
living at maturity, having many types of biochemical metabolism, thin cell walls, are usually isodiametric in shape, etc.
Collenchyma:
cells specialized in plastic support,
living at maturity, having limited metabolism, irregularly thickened cell walls, elongate in shape
Sclerenchyma:
cells specialized in rigid support,
dead at maturity, no metabolism, heavily thickened and lignified cell walls, elongate in shape
Your instructor may walk you through several stages in cell division to remind you of this physiological process. A standard slide (Allium (onion) root tip) may be used to show the various stages. Collenchyma may be observed in freehand sections of Apium (celery) or young stems of beans, etc.
What do I hand in?