Download onion cell (before)

ONION OSMOSIS LAB
Water passes through aquaporins in cell membranes from an area of high water concentration (low
solute concentration) to an area of low water concentration (high solute concentration). This process is
called osmosis. It requires no cellular energy to be used, and occurs due to the random, continuous
motion of all molecules.
If a cell is placed in an environment in which the concentration of water is less / concentration of
solutes is greater than in the cell (hypertonic), water will flow from the cytoplasm and/or water vacuole
through the membrane into the environment. (Some solute moves into the cell but this does not account
for the dramatic changes that you see or weigh) The cytoplasm and/or central vacuole of the cell shrinks.
In an animal cell, the entire cell shrivels. In a plant cell, the central cacuole shrinks away from the cell
wall. A cell placed in a hypotonic environment, with a higher concentration of water / lower
concentration of solutes, will gain water by osmosis. A cell placed in an isotonic environment, containing
the same concentration of water and solute as in the cell, will not gain weight, as water moves equally in
both directions.
Purpose:
to observe the process of osmosis in living cells.
Materials:
red onion cell, slides, coverslip, compound microscope, 4% salt solution, distilled water,
Procedure:
1. Make a wet mount of a small, thin piece of purple epidermis from a red onion bulb's leaf scales.
2. Photograph and label what you observe under high power. Be sure to include: cell wall (note:cell
membrane not visible since it adheres tightly to cell wall), nucleus, central vacuole (takes up most of the
space in the cell and contains water and a reddish/ purple pigment called anthocyanin, central vacuole
membrane - will only be visible after adding salt).
3. Lift off the coverslip and place 2 drops of 4% or 8% salt solution directly on the sample.
4. Photograph your cells. Note the color and position/shape of the vacuole.
Label central vacuole membrane.
5. Lift the coverslip and add 3-5 drops of distilled water to the slide.
6. Photograph the cells. Again, note the color and shape of the vacuole.
7. Watch onion cell plasmolysis at: https://www.youtube.com/watch?v=rMtaqq2bmFU
Results:
onion cell (before)
onion cell – after % salt / % water
onion cell – after distilled water (100% water)
Image Analysis:
1. Download the software program Image J at: http://rsb.info.nih.gov/ij/download.html
Download ImageJ for Mac OS X from the Download page. The ZIP file you download (Image1.xx.zip) should automatically
expand to a folder named "ImageJ". Copy this folder to the Applications folder, and open it. The first time you run ImageJ you
may get get an "ImageJ can't be opened because it is from an unidentified developer" message, which can usually be bypassed
by right clicking on ImageJ.app and selecting "Open" from the drop down menu. if that doesn't work, open the "Security &
Privacy" panel in System Preferences and change "Allow apps downloaded from:" to "Anywhere". You can switch back to the
original setting once ImageJ is running.
2. In Image J, open your microscope photo of the red onion cell "before". Convert your image to a black
and white image: Image / Type / 32 bit.
3. Pick 3 representative cells and measure the average pixel (color) value in those cells by using the
freehand tool to outline the central vacuole. Select Analyze / Measure and read the mean pixel value.
(lighter = higher number; darker = lower number range = 0-256)
freehand tool
outlined central vacuole
mean
pixel value
nucleus
central
vacuole
filled with
water and
pigment
cell
wall
4. Open your red onion "after" image. Convert it to a 32-bit black and white image. Measure the
area of 3 cells by outlining each with the freehand drawing tool. Measure along the inner portion of
the cell wall. We are assuming that the central vacuole has almost the same area as the entire cell.
Analyze / Measure
freehand tool
yellow outlined cell
5. Measure the area of the central vacuole of those same 3 cells using the freehand selection tool.
Analyze / Measure
6. Measure the average pixel value of the central vacuole in those cells by reading the Mean value in
the results window.
Table 1 Data and statistics for onion osmosis in X % salt solution
% salt
average pixel
average pixel
area
value of central value of
cell/central
vacuole before
central
vacuole before
vacuole after
cell 1
cell 2
cell 3
avg
stand.dev
95% CI
area central
vacuole after
% change
in area
7. Get the results from another group that used a different % salt than your group.
Table 2 Data and statistics for onion osmosis in X % salt solution
% salt
average pixel
average pixel
area
value of central value of
cell/central
vacuole before
central
vacuole before
vacuole after
cell 1
cell 2
cell 3
avg
stand.dev
95% CI
area central
vacuole after
t-test
Was there a difference in the effects of the different salt solutions? Do a t-test to find out!
null hypothesis =
p value =
Explain these results.
% change
in area
Discussion Questions:
1. Under each picture in the caption, explain the changes in the size of the central vacuole and relative
color of the pigment (mean pixel value) following the addition of the salt solution and then the distilled
water. Use arrows and labels showing the movement of water.
2. Animal cells exposed to distilled water swell and burst but plant cells do not. Based on your
knowledge of cell structure, why might this occur?
3. HONORS ONLY: How do organisms that live in fresh or salt water cope with an environment that
differs from that of their cells? Use your class notes, textbook, Haiku Osmoregulation article and/or this
website (or others) to help you answer this. http://en.wikipedia.org/wiki/Osmoregulation
4. The solute concentration of contact lens saline solutions must be isotonic to the cells of your eyes.
Why?
5. How should sports drinks need to be formulated so that they are able to effectively rehydrate your
cells? Why might Coke or lemonade not be good at quenching your thirst?