BIOL 183 LAB 1

CELL BEHAVIOR AND MEMBRANE

PERMEABILITY I

OVERVIEW
This investigation introduces you to methods of microscopy, and uses plant (Elodea) and
animal (erythrocyte) cells to investigate the permeability of plasma membranes. Some
goals of this lab investigation are:
Learn how to use a compound microscope to observe wet mounts of cells, using phase
contrast and bright field microscopy.
Understand how isosmotic, hyperosmotic and hypoosmotic conditions affect both
plant and animal cells

PRE-LAB ASSIGNMENT
In addition to this lab, read Campbell, pp. 125-126, Concepts 7.2, 7.3, and 7.4.
Complete the microscope tutorial at this website (click on microscope tutorial):
http://virtualurchin.stanford.edu/microtutorial.htm

INTRODUCTION
The cells of organisms and the molecules they contain are too small to be studied
directly with our eyes. Instead, we use a variety of microscopes to gain information about
cells and their contents. With the light microscope, the image of a specimen is formed by
passing focused light through it and then refocusing and magnifying with a system of
lenses. The compound light microscope you will use in todays laboratory should allow
you to identify and examine the nucleus, cytoplasm, chloroplasts, cell wall, and maybe
even vacuoles of plant cells, but the plasma membrane, normally pressed against the
cell wall in plant cells, will remain invisible.

Microscopy
A compound light microscope has three lenses (or lens systems). The condenser
lens is under the stage and focuses the light that will pass through the specimen. The
objective lenses are nearest the object or specimen being viewed, and form a magnified
image of the specimen, and the ocular lenses are nearest the viewer's eye, which further
magnifies the image.
Magnification, the most obvious feature of a microscope, is a measure of how big
the specimen looks to your eye compared to life size. Magnification is usually written
as a number followed by an X, which stands for times life size (10X then, means the
specimen appears ten times as large as life size).
Resolution (or resolving power) is the limit to which small objects may be seen as
separate objects. The resolution of the human eye, for instance, is about 100
CELLS & PLASMA MEMBRANES I 2

micrometers. A micrometer (m) is 0.001 of a meter; we can distinguish two dots as

being separate from one another when they are as little as 0.1mm apart. Plant and animal
cells are typically 10-100 m in size, so they are not visible unless magnified under a
microscope. Increased magnification is of no use without increased resolution. Without
the ability to distinguish detail, a fuzzy image will be magnified only to produce a larger
fuzzy image. The resolution of a microscope is limited by the diffraction, or scattering, of
light as it passes through the specimen and the lens. Diffraction decreases however (and
resolution improves), by using an illumination source of shorter wavelength than visible
light. The electron microscope uses a beam of electrons (much shorter wavelengths)
instead of light to pass through the specimen, and therefore has a much higher resolving
power.
Contrast is another important feature of the light microscope. Even with the
proper magnification and resolution, contrast is necessary in order to distinguish the
specimen from its background (or else it will be like watching a polar bear in a
snowstorm). Contrast arises from the different ways in which light passes through
various parts of the specimen.
Brightfield microscopy is the most basic technique, in which the sample is
visualized simply by illumination of white light. However this method does not provide
enough contrast for samples that are transparent, such as unstained cells or non-
pigmented tissue. For these specimens, its necessary to use stains or phase contrast.
Phase contrast is a technique that exploits the slight differences in refractive index parts
of the specimen and the microscope slide. Light that has traveled through the sample has
a wavelength that is slightly out of phase with light that has not.

The Plasma Membrane

The plasma membrane, a (primarily) lipid-protein barrier that surrounds the cell,
controls the movement of molecules into and out of the cell. It has been observed that
different substances penetrate the cell at different rates and that some substances do not
penetrate at all, thus the plasma membrane is referred to as selectively permeable. In
many cases, solute molecules (the dissolved substance) could be blocked, but molecules
of the solvent (the liquid in which the solute is dissolved) are allowed through. The
solvent of biological systems is water, and the movement or diffusion of water (caused by
a solute concentration difference) through a selectively permeable membrane is called
osmosis.
Osmosis is a special form of diffusion. It is the diffusion of water across a semi-
permeable membrane (such as a plasma membrane) from a region of high water
concentration to one where water concentration is lower. Because osmosis involves the
movement of water, it is the concentration of water molecules in a solution that
determines the direction of water movement. For example, one liter of a 1.0 M solution
of NaCl has a lower concentration of water than does one liter of a 0.5 M solution of NaCl.
If these two solutions were separated with a membrane permeable only to water, in
which direction would the water move? The greater the difference in concentration
between two solutions, the greater is the tendency of the water to move. The number of
particles in the solution causes the concentration difference, not the kind of particle. If a
cell is in a solution that contains more dissolved particles than found inside the cell, the
solution is called hyperosmotic. A cell in a solution of relatively fewer solute particles is
in a hypoosmotic environment. An isoosmotic environment contains the same number
of solute particles as the cell. Tonicity, on the other hand, refers to the osmotic behavior
of a cell in an environment. A cell loses water in a hypertonic environment, gains water in
CELLS & PLASMA MEMBRANES I 3

a hypotonic environment, and has no net flow of water in an isotonic environment. (Tonicity
and osmolarity does not always mean the same thing. As we will see in next weeks lab,
an environment could start out as isosmotic to cells, but not be isotonic.)
Several different mechanisms are involved in the transport of substances across the
plasma membrane, depending on whether the substances cross down the concentration
gradient, or against the concentration gradient. Diffusion and osmosis are the simplest
forms of transport; they occur down the concentration gradient and are caused by purely
physical and chemical forces. Therefore, osmosis and diffusion are cases of passive
transport; the cell does not have to expend any energy for a substance to move across a
membrane by passive transport. Even though this type of transport is passive, the effects
of passive transport are actively controlled and regulated by cells and organisms. If cells
fail to control diffusion, they may lose valuable chemical compounds they have made at
great expense or they may become flooded by an influx of harmful substances from the
environment.

What would happen if cells failed to control osmosis?

?
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METHODS AND MATERIALS

Care and Handling of Microscopes
Grasp the microscope firmly by the recessed handle (top rear of the column) with one
hand and put your other hand underneath the base of the microscope. Always use
two hands to remove a microscope from its cabinet, to carry it anywhere in the room,
and to return it to its cabinet. Keep the instrument upright. These precautions are
designed to eliminate accidental dropping and to reduce the chance that the ocular
lenses will accidentally fall to the floor and break.

Clean the ocular and objective lenses when necessary. Students often have a difficult
time viewing a specimen because one or more lenses are dirty. Usually you can
deposit enough moisture on the lens by breathing heavily on it, and then polishing
the lens with lens paper.

The adjustable knobs and moveable parts of the microscope should be used, moved,
and adjusted. However, do not apply full force to any adjustment; you will only strip
threads, break cast aluminum parts, or cause the axes to become misaligned. Tell
your Instructor or Teaching Assistant if you have not been able to correct a problem
with your instrument. Remember, a microscope is a precision instrument, and is easily
damaged.
Parts of a Microscope
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Look at your microscope and find all of these parts:

Light Switch Control: The light switch and brightness controls are on the right side of
the microscope, above the focus knobs. Youll find you will need less light on low power
magnification and brighter light on higher power magnification. Use only as much light
as necessary to illuminate the specimen.
Light Source: On our microscope the light source is built into the base and is directly
under the condenser.
Condenser: A lens system under the stage that gathers light from the light source and
focuses it on the specimen. There is an iris diaphragm in one part of the condenser that
can be adjusted to allow the viewer to see different parts of the cell when using bright
field illumination. You should experiment with this control (a lever in the front of the
condenser).
Condenser Adjustment Control: Under the stage on the left side of the condenser is a
small knob that is used to adjust the height of the condenser.
Ocular: The piece you look through. Sometimes called an ocular lens or eyepiece, this
unit is really a series of lenses. Our microscopes are binocular, having two oculars.
Learn to use both eyes. Note that the left ocular has its own knurled focus ring. To
properly focus both oculars for your eyes, first look only through the right ocular and
focus on the specimen with the microscopes focus knob. Then look at the specimen
through only the left ocular and focus with that oculars focus ring. You should adjust the
width of the oculars to match the width of your eyes! The ocular lenses have 10X
magnification.
Objective lens: This series of lenses directly above the specimen serve to magnify and
focus the image. These scopes have three bright field objectives (4X, 10X, and 40X) and
one oil-immersion objective (100X). You can see the magnification written on each lens
tube.
Note that the total magnification will be the magnification of the ocular lens multiplied
by the magnification of the objective lens. If youre using, for example, the 40X objective
lens, the total magnification with which youre viewing the specimen is 400X.
Nosepiece: The rotating turret to which objectives are mounted. There are preset
positions for each objective, detected by slight pressure changes while turning the
nosepiece and usually a clicking noise. You should not grab the objectives to turn the
nosepiece use the black ring instead.
Stage: The flat surface upon which slides are placed. The slide is moved left/right and
front/back in its holder clamp by two knobs projecting downward from the stage (on
the right).
Adjustment (Focus) Knobs: Both coarse (large) and fine (small, inner) adjustment knobs
are found on both sides of our microscopes. Remember that the coarse adjustment is
used only with the low-power (4X, 10X) objectives. The fine focus is used to hone in on
the image. These knobs control a gear mechanism that raises and lowers the stage.
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A. Operating the microscope

Unless care is taken to insure proper illumination and contrast, even a research
quality instrument will yield mediocre results, and an adequately magnified image will
be only moderately well resolved!
1. Turn on the lamp, and adjust the brightness with the light intensity knob.
2. Turn the revolving nosepiece to engage the 10X objective. Make sure the nosepiece
stops with an audible click.
3. Insert a letter e slide into the stage clip under the 10X objective, center the specimen
with the position controls, and bring the specimen into focus. Make sure both oculars
are focused: Looking through the right eyepiece with your right eye, turn the coarse
focus knob to bring the specimen into focus. Use the fine focus knob to make fine
adjustments. Looking through the left eyepiece with your left eye, turn the
adjustment ring to focus the specimen.
4. Adjust the interpupillary distance of the eyepieces.
5. Adjust the brightness if necessary, readjust focus if necessary, and adjust the iris
diaphragm to provide enough contrast.
6. Slowly swing the high-power (40X) objective into position, making sure it doesn't hit
the slide on the stage. Most modern microscopes are parfocal; once the image is
brought into sharp focus under low power, it will remain in focus when the high
power objective is swung in place. (However, a small fine focus adjustment may be
necessary--but DON'T USE COARSE FOCUS!) How does switching to high power affect
the brightness of the field? (Adjust the iris diaphragm if necessary!)
7. Return to low power and note the orientation of the image. Does the letter appear
normal or upside down? Move the slide to the left. Which way does the image move?
Lower the stage and return the slide to the appropriate tray.

B. Make wet mounts and observe cell structure

Observing plant cells
1. Obtain a clean microscope slide and cover slip. Pick a relatively small, young leaf
from the tip of a shoot of Elodea. Place it on a microscope slide in a drop or two of
aquarium water with the top side up.
2. Make sure the leaf on the slide is flat and free of wrinkles. Lower one edge of the
cover slip to the slide so that it touches one side of the drop of water at about a 45
angle (see below).

3. After the water has spread across the entire edge of the cover slip, carefully lower
the rest of the cover slip in order to avoid trapping air bubbles under the cover slip
(air bubbles appear as round shapes with heavy dark outlines under the
microscope).
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4. Excess water can be soaked up by carefully placing a tissue along the edge of the
cover slip. If your slide appears to be drying out while observing it, add a drop of
water to the edge of the cover slip.
5. Under low power (using the 10x objective), find a group of cells near the midrib and
toward the base of the leaf. Once the specimen is in focus, adjust the light intensity
and condenser so that the cells are clearly visible. Switch to high power (40x
objective); you will probably find it necessary to readjust the iris diaphragm
(increasing the amount of light) for optimum visibility under high power.
Remember that the cells have depth; how many layers are distinguishable? Try to
finely focus up and down on an individual cell in one layer to explore its structure.
Examine and identify the cell wall, nucleus, chloroplasts, cytoplasm, and central
vacuole.

Make a
drawing
of what
you see:

NOTE: A large central vacuole is characteristic of most mature plant cells, in which case it
occupies the vast majority of cellular area, pushing the cytoplasm (including chloroplasts) and
nucleus to the periphery of the cell, just inside the cell wall. It may be hard to see the vacuole.

Observe animal cells

A good example of a typical animal cell can be sampled from the inner surface of your
cheek, inside your mouth. These cheek cells, squamous epithelial cells, are constantly
sloughing off and being replaced, so you can remove some of these cells without harm.
Use a toothpick to lightly scrape the inside of your cheek. Stir the scraped material in a
drop of physiological saline solution (PSS) on a microscope slide. Add a coverslip. Stain
your specimen by adding a drop of stain (e.g., methylene blue or toluidine blue) to one
side of the coverslip and draw the stain through by touching a tissue to the other side of
the coverslip. Observe under the microsope with bright field optics (low and then high
power).
What organelles can you see? How has the stain helped (or hindered) your view of cell
details?

Make a
drawing
of what
you see:

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C. Membrane Function: Observing the effect of solute concentration on

cell structure
Osmosis across plant cell plasma membranes
1. Prepare a wet mount of another Elodea leaf in a drop of 3X PSS solution (with
another clean slide and cover slip). Make a drawing of what you see (below). Pay
particular attention to the location and distribution of the chloroplasts; this will tell
you whether the vacuole continues to press the cytoplasm to the periphery or not.

Describe what happens to the cells, comparing this Elodea wet

? mount to the first one (in aquarium water):

2. Now prepare a wet mount of another Elodea leaf in a drop of distilled water (with
another clean slide and coverslip).

Describe what happens to the cells, comparing this Elodea wet

? mount to the previous ones:

Elodea cells in 3X PSS: Elodea cells in dist. water:

Make a
drawing
of what
you see:

Construct an explanation for what you just observed:

? (Some questions to consider: Do you think that the plasma membrane of Elodea
cells is selectively permeable? If so, can water diffuse through? Can salt?)

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Osmosis across animal cell plasma membranes

Now that youve seen what happens when plant cells are bathed in solutions of
varying concentrations, lets examine what happens to animal cells. Since the surface coat
of most animal cells is thin, flexible, and stretchable (to a limited extent), osmotic influx
or efflux of water can cause swelling and shrinking.

Mature red blood cells (erythrocytes) are nothing more than packages of
hemoglobin (mammalian erythrocytes dont even have nuclei when mature) bound by a
plasma membrane permeable to small molecules, such as oxygen and carbon dioxide, but
impermeable to larger molecules, such as proteins, sodium chloride, and sucrose. The
plasma membrane of erythrocytes is also permeable to water. You will observe what
happens to mammalian red blood cells when placed in solutions of various solute
concentrations: PSS, 3X PSS, and distilled water.

Construct a hypothesis and prediction for the behavior of red

Hypothesis
blood cells when placed in these 3 solutions.

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Experimental Protocol
Use a transfer pipette to place a drop of red blood cell suspension on a clean
microscope slide, and add a coverslip.
Observe the slide at low (10 X objective; 100X total) and high (40X objective; 400X
total) magnification with a compound microscope. (You may have to make your
observations near the edge of the coverslip where the cells are not as densely
packed.)
Record your observations in Table 1 (next page).

Red blood cells in physiological saline solution (PSS):

Make a preparation of red blood cells in mammalian Ringer's solution (PSS) by
using a transfer pipette to add two drops of blood to a small test tube. Immediately
add 4 ml of PSS to the tube. Cover the tube with a small piece of Parafilm and invert
to mix.
Note the appearance of the suspension in the tube -- is opaque or transparent? Hold
the tube up in front of this page can you clearly read the words through the
suspension in the tube?
Remove a drop of this suspension, prepare a wet mount, and observe the cells with a
compound microscope. Make a quick sketch of the cells.
Record your observations in Table 1 (next page).

Red blood cells in 3X PSS:

Make a preparation of red blood cells in salt solution that is greater than
physiological saline (3X PSS) by using a transfer pipette to add two drops of blood to
a small test tube. Add 4 ml of 3X PSS to the tube, cover with Parafilm, and mix.
Note the appearance of the suspension. Hold the tube up in front of this page can
you clearly read the words through the suspension in the tube?
Prepare a wet mount, and observe the cells with a compound microscope. Make a
quick sketch of the cells and describe the process that has occurred.
Record your observations in Table 1 (next page).

Red blood cells in distilled water:

Make a preparation of red blood cells in distilled water by using a transfer pipette to
add two drops of blood to a small test tube. Add 4 ml of distilled water to the tube,
cover with Parafilm, and mix.
Note the appearance of the suspension. Hold the tube up in front of this page can
you clearly read the words through the suspension in the tube?
Prepare a wet mount, and observe the cells with a compound microscope. (Why are
the cells not as easily visible?) Make a quick sketch of the cells and describe the
process that has occurred.
Record your observations in Table 1 (next page).
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Table 1. Effect of solutions of various osmolarities on bovine red blood cells.

Solution Shape of Cells

Red blood cells
(RBC) only

RBC + PSS

RBC + 3X PSS

RBC + dH2O

Explain your results in terms of your hypothesis:

1. Based on your hypothesis, predictions, and results, which of the three solutions is
hyperosmotic to bovine red blood cells? __________

Which is hypoosmotic? ___________

Which is isosmotic? __________

2. What has happened in each case, and why? (Include evidence from both
microscopic examination and observations of the transparency of the solutions.)

3. PSS is isosmotic to red blood cells. The total osmolarity inside RBCs is 0.3 osmoles, yet
PSS is a 0.15 M NaCl solution.
a. Explain why 0.15 M NaCl is isosmotic to something that contains 0.3 total
osmoles.

b. What molarity of glucose would be isosmotic to RBCs? Explain your answer.

4. When a person is severely dehydrated they receive physiological saline solution

(PSS) intravenously. Why arent they given intravenous water alone to reverse
dehydration?

Post-Lab Assignment
As a group, transfer your answers above to the worksheet handout you received. Hand
in one worksheet per group. Be sure to put all names of group members on the
worksheet. Your lab instructor will discuss the pre-lab assignment for next weeks lab
(which will be posted to your Moodle Lab site).