Exercise 2:
Cell Membranes
Related reading in text: p 122-127, p147-150; Handout
Objectives:
ƒ
ƒ
To examine cell response to changing osmotic conditions.
To examine the effect of detergents on cell membranes.
Part 1. Cell response to changing osmotic conditions
Changes to plant cells
Make the following Elodea slides: (remember to use a single, young leaf)
1- Elodea + 1 drop of water + coverslip
2- Elodea + 1 drop of 0.9% saline + coverslip
3- Elodea + 1 drop of 10% saline + coverslip
Observe and sketch each at 5X and 10X. Label cell wall, cell membrane, vacuole, chloroplast and
nucleus (if observed). Describe what happened in each condition.
Which solution was isotonic? Hypertonic? Hypotonic?
Changes to animal cells
Obtain a clean slide. Add 1 drop of 0.9% saline, 1 drop of sheep’s blood, and then a coverslip.
Observe under 5X and 10X. Sketch the red blood cells.
Repeat the procedure with 10% saline and distilled water. (Note: For best results, prepare the slide
just before you observe it.)
Red blood cells in a hypertonic solution will lose water and shrink or crenate. Which of the
solutions above was hypertonic?
Red blood cells in a hypotonic solution will take in water and swell (hemolysis). Eventually the
cell membrane may be come leaky or even burst. Which of the solutions above was hypotonic?
Did you observe a nucleus in any of the red blood cells? Why or why not?
Obtain 3 clean test tubes. Set the tubes up as follows
:
1- 2 ml distilled water
2- 2 ml 0.9% saline
3- 2 ml 10% saline
Add 100 µl of blood to each of the test tubes. Mix gently. Observe the tubes and note the results
in the table below (copy into your notebook!). Hint: Look for changes in turbidity (cloudiness)
in the tube by holding your lab protocol sheet behind the tube.
Tube
1
2
3
Condition
water
0.9% saline
10% saline
Appearance (clear/cloudy)
Which of the tubes had the right conditions for hemolysis of the red blood cells?
What did the tube look like?
Part 2. Disrupting membranes
When studying cells, we may want to lyse the cell and collect the subcellular organelles, nucleic
acids and proteins; or we may be interested in studying the cytoskeleton itself. Either way, we
need to get rid of the cell membrane.
One of the components of the cell membrane is the lipid bilayer. Lipids are nonpolar and will not
dissolve in water (good thing for us since our cells are constantly surrounded by water). Lipids
will dissolve in a nonpolar solvent but many nonpolar solvents are toxic and not a good choice.
Detergents, however, are the perfect choice. They have both a polar and nonpolar end. The polar
end of the detergent molecule lets them dissolve readily in water; the nonpolar end of the detergent
molecule will bind to lipids and pull them away. In the following activity, you will make some
solutions commonly used in cell biology protocols and test their effectiveness at disrupting red
blood cell membranes. Solution preparation: Prepare a 1 % solution of each of the following
reagents:
1.
2.
3.
4.
Triton X, EDTA, SDS, and Palmolive. (You should prepare 10 ml of each solution using
0.9% saline as your solvent. Why do you use saline and not plain water?)
Solution Testing: Number 5 small test tubes 1-5. Use the chart below and add 2ml of test
solution to each tube. Then add 100 µl of blood to each tube.
Mix each tube gently.
Record your results in the chart below (and in your notebook).
Test Solution
Clear/cloudy?
Cells Lysed?
Tube #
______________________________________________________________________________
1
0.9% saline
______________________________________________________________________________
2
Triton X
______________________________________________________________________________
3
EDTA
______________________________________________________________________________
4
SDS
______________________________________________________________________________
5
Palmolive
______________________________________________________________________________
Did all of the solutions you tested lyse the cells? Explain the results.
Cleaning Agents
Cleaning clothes, dishes and hair would be easy if all soils and stains dissolved in water-washing
would consist of a simple rinse. But solvents such as water are finicky liquids that dissolve some
chemicals better than others. It's a matter of attraction: if solvent molecules are more strongly
attracted to one another than to a sod molecule, they will have trouble dissolving it. Unfortunately,
many of the soils we wane to remove don't bind well to water.
Water is an excellent solvent for polar chemicals-including salts, which dissociate easily into
electrically charged ions, or sugars, which have charged regions. Water itself is a polar molecule;
its hydrogen atoms are slightly positive, its oxygen atom slightly negative. When water molecules
encounter a polar soil molecule, electrostatic attraction causes them to entrap the molecule and
carry it away. But nonpolar chemicals such as oils and fats have no charged regions with which to
attract water. These soils can be dissolved in nonpolar solvents-including
SOAPS
Most soaps are salts derived from fats or oils and consist of positively charged sodium ions and
negatively charged molecular chains. Each negative ion's charge is located at one end, where its
nonpolar hydrocarbon chain ends in a polar carboxylate group. When you add soap to water, its
sodium ions dissolve, and the now negatively charged chains form micelles. The chains also coat
the surface of water molecules, reducing their surface tension and allowing them to penetrate
fabrics.
DETERGENTS
Unfortunately, soap works poorly in hard water. The positively charged calcium, magnesium and
iron ions in hard water
BLEACHES
Some stains, like ink spots, are bound so tightly in place that they can't be dissolved and must be
destroyed instead. Their colors are often associated with weakly bound electrons, such as those
involved in double bonds between atoms. Bleaches attack those vulnerable electrons and use
electron-withdraw- 7ng,atoms-such as oxygen and chlorine-to snap them up. The stain
molecules then become colorless and invisible.
BRIGHTENERS
As they age, white fabrics acquire a yellowish cast because they begin to absorb light at the blue
end of the spectrum instead of reflecting it. To replace this "missing" blue light, brighteners are
added to many detergents. These fluorescent dyes absorb invisible ultraviolet light and use its
energy to emit blue light. This extra blue hides the fabric's yellowed appearance. When exposed to
sunlight, brightened fabric has a strong bluish glow and appears brilliantly white. We are so used
to this glow that nearly, all white fabric is predyed with brighteners to make it look white enough
for our tastes.
__________________________________________________________________
DID YOU KNOW
When wet, hair and many fabrics acquire a weak negative charge. This charge gently
repels both the negatively charged soap and detergent nacelles and keeps them from redepositing
greasy soil molecules. But the molecular ions in most conditioners and fabric softeners have
positively charged ends that attract them to hair and fabric, causing them to remain there as the
water evaporates. They then release their softening or hydrating molecules
It's hard to combine shampoo and conditioner in a single bottle because the negatively
charged shampoo ions and the positively charged conditioner ions tend to interfere with one
another. 'the hair cleaners that contain both ingredients trap the conditioner molecules in
crystalline shells or complexes that open only when exposed to excess water. So the conditioner
molecules are hidden while you're lathering your hair but are released when you rinse
Many fibers carry polar chemical groups to which water molecules bind tightly, making
them swell and stretch when wet. As those fibers dry, they return to their original sizes but not
their original shapes. The result is structural damage to the garment. To avoid such damage, these
fabrics can be dry-cleaned with nonpolar, albeit toxic, solvents such as perchloroethylene.
Detergents added to these solvents form inverse micelles that can dissolve polar soils.
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LOUIS A. BLOOMFIELD is a professor of physics at the University of Virginia
and author of How Things Work: The Physics of Everyday Life