A&P 242 Lab 1: Review: Membrane Transport and Micrscope Use
Passive Transport: Diffusion, Osmosis and Transport Review
Molecules in air and molecules in liquids are in motion due to kinetic energy. The movement is random and
is called Brownian motion. As a result of this random movement, with time, molecules tend to become
evenly mixed (diffusion of fragrance from perfume, tea solutes). When you put food coloring in a glass
of water, the molecules move as a result of kinetic energy, spreading out to distribute evenly in the
water. The term that describes such unlimited mixing is diffusion.
Diffusion happens in our bodies and is the force that moves substances across cell membranes (passive
diffusion). Cells are surrounded by a membrane that is a barrier separating inside from the outside and
prevents the free diffusion of all solutes except for water and small, nonpolar substances.
In the normal situation, the concentration of solutes inside the cell equals the concentration of solutes
in solution outside of the cell even though the solution composition is different (due to the actions of
ATP dependent pumps).
Check your understanding of osmosis: If the number of dissolved particles (solutes) inside a cell is
higher than that in the solution outside the cell, the cell contents are said to be hypertonic to (more
concentrated than) the environment. If the cell contents are more dilute than the environment, the cell
is hypotonic to the environment. When both are balanced, the concentrations of the cell and its
surroundings are isotonic.
When the cytoplasm and extracellular fluids have different total # of solutes dissolved in them, water
will pass through the plasma membrane in a process called osmosis, down its concentration gradient so
that the two regions will become more similar in concentration. A cell whose contents were hypertonic
to the environment would gain water; one that was hypotonic to the environment would lose water.
Isotonic: The solutions being compared have equal concentration of solutes.
Hypertonic: The solution with the higher concentration of solutes.
Hypotonic: The solution with the lower concentration of solutes.
EX: Draw a line on the graph to show the relationship between osmolarity of a solution and solute
concentration.
EX: Check your understanding of Diffusion:
a. Draw a line of the plot that shows the relationship between temperature and diffusion rate.
b. Draw a second plot (dotted line) to show the relationship between molecule size and diffusion
rate (you’ll need to add size to the X axis)
If you need more of a review, go to this webpage and try to answer the questions like the one shown
below: http://www.vivo.colostate.edu/hbooks/cmb/cells/pmemb/osmosis.html
Glucose has an atomic mass: 180, Lactose: 342. Can you explain how the solutions were made?
Example 1: Glucose is a monosaccharide and sucrose (table sugar) is a disaccharide.
Example 2: NaCl or sodium chloride is, of course, table salt. Before doing the problem, think about what happens to
salt when it is dissolved in water. *
NaCl
Na+ + Cl-
Exploring Membrane Potentials (The Bean Lab)* Courtesy of Dr. Dee Silverthorn, University of Texas,
Austin
Open channel
Cell membrane
Closed channel
ECF
ICF
This exercise is designed to help you understand the basis of resting membrane potentials, graded potentials, and
action potentials in living cells. You will use different kinds of beans to represent different ions. A piece of paper
divided into two sections represents the intracellular (ICF) and extracellular (ECF) compartments. The two
compartments are the same size. The dividing line between the compartments represents the cell membrane, and
colored rectangles of paper are used to indicate ion channels. When the long axis of a channel is perpendicular to
the membrane, the channel is open and ions can flow through it.
Electricity Review
Atoms, the smallest units of matter, are electrically neutral entities. They are composed of positively charged
protons, negatively charged electrons, and uncharged neutrons, but in balanced proportions so that an atom is
neither positive nor negative. The removal or addition of electrons to an atom creates a charged particle known as
an ion. Positive ions, or cations, that are important in the human body include Na+, K+, Ca++ , Mg++ and H+. For each of
these positive ions, somewhere there is a matching electron, usually found as part of a negative ion, or anion. For
example, when Na+ in the body enters in the form of NaCl, the "missing" electron from Na + can be found on the Cl-.
The following principles are important to remember when dealing with electricity in physiological systems:
(1) The Law of Conservation of Electric Charge states that the net amount of electric charge produced in any
process is zero. This means that for every positive charge on an ion, there is an electron on another ion.
Overall, the human body is electrically neutral.
(2) Opposite charges (+/- ) are attracted to each other, but two charges of the same type (+/+ or -/- ) repel. With
ions, the ion species does not matter: only the net charge on the ion is important. Thus a Cl- has one negative
charge that is equal and opposite to one Na+ or one K+.
(3) To separate positive and negative charges, it is necessary to use energy. For example, energy is needed to
separate the protons and electrons of an atom.
(4) If separated positive and negative charges can move freely toward each other, the material through which they
are moving is called a conductor. Water is a good conductor of electricity. But if separated charges are unable
to move through the material that separates them, the material is known as an insulator. The phospholipid
bilayer of the cell membrane is a good insulator, as is the plastic coating on electrical wires.
In each of the following exercises, you will distribute ions (beans) between the two compartments, set the
permeability of the dividing membrane by opening or closing a channel, then predict the movement of the ions
between the compartments based on their electrochemical gradients. Write in the answers to the questions as you
work.
Exercise 1: What happens if a membrane is freely permeable to all ions?
Place 10 Na+ and 10 Cl- ions in the ECF compartment (ECF = Extracellular Fluid). Place 10 K+ and 10 protein anions
(A- ) in the ICF compartment (ICF = Interstitial Fluid or Cytoplasm). Because the compartments are the same size,
equal numbers of ions (equal amounts) in the two compartments are the same as equal concentrations.
(Q1) Is the ECF electrically neutral? _______ Is the ICF electrically neutral? ________
(Q2) Is there an electrical gradient that would move ions from one compartment to the other? ________
(Q3) Assume that the membrane is freely permeable to all four types of ions. Is there a concentration gradient
for Na+ ? __________ for K+ ? __________ for Cl- ? __________ for A- ? __________
Rearrange the ions so that any concentration gradients are abolished and the system has come to equilibrium. In
the space below, draw in the system at equilibrium.
ECF
ICF
Without a selectively permeable membrane around cells, the body would be unable to create or maintain the ion
gradients needed for electrical signaling.
Exercise 2: What happens when a membrane is selectively permeable, allowing only certain ions to cross?
Molecules that cannot diffuse freely through the phospholipid bilayer of the cell membrane require protein
transporters. – or “doors”. Ion channel proteins are water-filled passageways that link the intracellular and
extracellular compartments. Ion channels therefore provide a pathway for ions and water to move rapidly between
the ECF and ICF. NOTE: Ion channels may restrict ion movement. For example, an ion may be too large or not the
right electrical charge to fit through the pore. For example, there are separate Na+ and K+ channels – that
discriminate on what can fit through the pore based on size.
LEAK CHANNELS VS> GATED ION CHANNELS: Some channels, called leak channels or pores, are always open,
allowing ions to move back and forth across the membrane. Another type of ion channel are the gated channels.
These channels spend most of their time closed. Special signals “gate” the channels, causing them to open. Some
of the signals that gate ion channels include: binding of chemical messenger molecules (chemically-gated channels),
changes in the electrical field across the cell membrane (voltage-gated channels), or physical changes such as
increased temperature or a stretch to the membrane and pops the channel open (mechanicall- gated channels).
From now on, assume that the cell membrane of your model is impermeable to all ions unless an ion channels for
that ion is placed on the membrane.
Rearrange the beans so that you again have 10 Na+ and 10 Cl- ions in the ECF compartment, and 10 K+ and 10 protein
anions (A- ) in the ICF compartment.
(Q4) Is the ECF electrically neutral? _______ Is the ICF electrically neutral? ________
(Q5) Is there an electrical gradient that would move ions from one compartment to the other? ________
(Q6) Are their concentration gradients for any ions? If so, which ones?
_____________________________________________________________________
Now place the rectangle representing the K+ leak channel on the membrane in the open position.
(Q7) Can K+ now cross the membrane? ________ In which direction will it move? _____
_____________________________________________________________________
(Q8) What force is moving the K+ ?__________________________________________
Move three K+ beans.
(Q9) Now what is the net electrical charge on the ECF? __________
(Q10) What is the net electrical charge on the ICF? __________
(Q11) Like charges repel and opposite charges attract. Why don’t the A - ions follow the K+?
Why don’t some of the Na+ move?
_____________________________________________________________________
On the number line below, mark and label the net charge for the ECF and the net charge for the ICF.
-------|--------|--------|--------|--------|--------|--------|--------|--------|--------|--------|------
-5
-4
-3
-2
-1
0
+1
+2
+3
+4
+5
When there is uneven distribution of ions between the intracellular and extracellular compartments, such as you
just created in your model, the electrical gradient between the two compartments is known as the “resting”
membrane potential difference, or membrane potential for short.
(1) The "resting" part of the name comes from the fact that this electrical gradient is seen in all living cells, even
those that appear to be without electrical activity.
(2) The "potential" part of the name comes from the fact that electrical and concentration gradients are sources
of stored or potential energy. When oppositely charged molecules move together, they release energy that can
be used to do work; in the same way, molecules moving down their concentration gradient can perform work.
The work done by electrical energy includes opening voltage-gated membrane channels , sending electrical
signals through nerve cells AND moving food across cell membranes (e.g. glucose).
(3) The "membrane potential difference" part of the name is to remind you that this number represents a
difference in the electrical charge inside and outside the cell. The word difference often gets dropped.
In your model, you created a membrane potential difference in which the ECF has a net charge of +3 and the ICF
has a net charge of -3. But in living systems, we cannot accurately measure the absolute number of electrical
charges on two sides of a membrane. So instead, we measure electrical gradients on a relative scale with a device
that measures the difference in electrical charge between two points. This device artificially sets the charge on
the extracellular fluid at zero millivolts (mV, this is known as the ground) and measures charge on the intracellular
fluid relative to the ECF (0 mV).
(Q12) Go back to the number line above. If you shift your mark for the ECF to zero, what is the new value for the
ICF charge?
_____________________________________________________________________
In biological systems, the actual value for the resting membrane potential of living cells is usually in the range of 70 to -90 millivolts (mV), ICF negative relative to an ECF value of zero mV.
Exercise 3: What establishes the resting membrane potential of cells?
Look at your model, which now has 3 K+ in the ECF and 7 K+ in the ICF. The K+ moved out of the cell in response to a
concentration gradient. But K+ is an ion, so it must also obey electrical gradients.
(Q13) If the net charge inside the cell is now -3, in which direction would K+ move along its electrical gradient?
_____________________________________________________________________
The ion is faced with two opposing forces that act in different directions: K+ moves out of the cell according to its
concentration gradient, but back into the cell according to its electrical gradient. Which gradient will it obey? The
simple answer is that it will obey the gradient that is stronger. But how do we know which is stronger?
There is no simple answer to this, but if we know the concentration gradient, we can use a mathematical equation
known as the Nernst equation to calculate the electrical gradient that exactly opposes that particular
concentration gradient. The membrane potential that opposes a given concentration gradient of an ion is known as
the equilibrium potential or NERNST potential for that ion. For example, if the K+ concentration of the ICF is 200
mM and the K+ concentration of the ECF is 20 mM, the concentration gradient would tend to drive K + out of the cell
and into the ECF. Using the Nernst equation, we can determine that when the ICF has a membrane potential of -60
mV (relative to the ECF), the electrical forces pulling K + back into the cell are equal in magnitude but opposite in
direction to the concentration gradient pulling K + out of the cell. Thus, the equilibrium potential for that
concentration gradient of K+ is -60 mV. (see your lecture notes and class discussion for Nernst equation)
Based on what you have just learned, see if you can answer the following questions. Remember that the cell
contains protein anions (A-) that cannot leave the cell and that the ECF fluid contains anions such as Cl - that cannot
enter the cell. The only ions that can move across the membrane are the ones that have a pathway provided by a
leak channel.
(Q14) Suppose you add some KCl (electrically neutral) to the ECF so that the ECF concentration of K + goes from 20
mM to 25 mM. What happens to the concentration gradient driving K + out of the cell?
(Q15) When the amount of K+ leaving the cell changes, as it did in the previous question, does the resting membrane
potential of the cell become more positive or more negative? Is this hyperpolarization or depolarization?
Remember that the resting membrane potential of the cell refers to the potential difference in voltage across the
membrane (with the outside voltage always equal to 0 mV). Does the absolute value of the resting membrane
potential difference increase or decrease?
_____________________________________________________________________
From this model cell, you can begin to understand how changing the K + concentration of the ECF (which includes the
blood) alters the resting membrane potential of a cell. If the body’s homeostatic mechanisms for maintaining ECF
K+ within a narrow range fail, the resting membrane potential of cells will change. This can have serious
consequences for excitable cells, such as nerves and heart muscle.
Now, based on what you learned about equilibrium potentials, see if you can use your model to answer the following
question. Rearrange the beans so that you have 12 Na+ and 12 Cl- ions in the ECF compartment, and 12 K+ and 12
protein anions (A-) in the ICF compartment. Remove the K+ leak channel and replace it with a Na+ leak channel.
(Q16) Will Na+ move across the membrane, and if so, in which direction?_________
_____________________________________________________________________
(Q17) If Na+ moves, what happens to the electrical neutrality of the model?___________
_____________________________________________________________________
(Q18) Based on the above model, predict the sign (e.g., positive or negative) of the equilibrium potential for Na+.
(Remember, the equilibrium potential is the electrical charge inside the cell that would exactly oppose a
concentration gradient.)
_____________________________________________________________________
_____________________________________________________________________
(Q25) What is the resting membrane potential of this cell relative to the ECF: positive, negative, or zero?
_____________________________________________________________________
(Q26) Change the Na+ channel to the open position. Which way will Na+ move, and what forces are acting on it?
_____________________________________________________________________
Now move the ions in the appropriate direction.
(Q27) What happens to the membrane potential of the cell as Na + moves? Does it become more or less negative?
_____________________________________________________________________
Membrane
potential
0 mV
-70 mV
Open Na+ channel
Time
On the graph above, draw in what happens to the membrane potential as Na+ moves.
(Q28) If the membrane potential moves closer to zero mV, the cell is said to have depolarized. If the membrane
potential moves farther from zero mV, the cell is said to have hyperpolarized. Did this cell hyperpolarize or
depolarize?
_____________________________________________________________________
Gated channels can be opened by a signal e.g. when a chemical binds to them (chemically-gated channels) or when
the cell membrane is deformed due to pressure on the membrane (mechanically-gated channels). When the channel
opens, ions have a pathway across the membrane and there will be net ion movement if there is a concentration
gradient for the permeant ion.
Replace the Na+ channel with a gated Cl- channel in the CLOSED position. Assume that the equilibrium potential for
Cl- is - 120 mV and that the membrane potential of this cell is -60. Now open the gated Cl- channel.
(Q30) Will Cl- move? If so, in which direction?_________________________________
(Q31) What forces favor and oppose Cl- movement?
_____________________________________________________________________
On the graph below, draw in what happens to the membrane potential of the cell when the Cl - channel opens.
(Q32) Did the cell depolarize or hyperpolarize?__________________________________
Membrane
potential
0 mV
-70 mV
Open Cl- channel
Time
Quick Review of Microscopes
Compound microscopes: parts and proper care
Microscopes are expensive shared instruments and must be handled carefully and cared for properly in order to
function optimally. Read the information below before obtaining your microscope.
Carrying the scope: When carrying a microscope, use one hand to support the base, and the other to grasp
the arm. Hold it in close to your body so it is less likely to bump into obstacles as you move about.
Cleaning: Keeping the microscope clean is important. Microscope lenses can only be cleaned with special
lens paper (see you instructor) otherwise the very expensive lens will be damaged. For the base and
the stage, you may use damp paper towels or Kimwipes (small lab tissues). Ask you teacher for help if you
have questions.
Using the compound microscope: Focusing and Magnification
1.
Make sure that your microscope is plugged in, the light is working and set to a medium brightness, and that
the scanning (4X) objective lens is rotated into place (in the light path)
2. Raise the stage to its highest point.
3. Place a slide on the stage of the microscope with the coverslip side facing up. With a compound
microscope, specimens are mounted between 2 thin pieces of glass - a thicker slide on the bottom called
the “slide” and a thinner slide on top called a coverslip. Also note that the specimen must be thin enough
for light to pass through it. Most specimens we’ll examine are thinly sectioned (10 x 10 -6 m) and stained
with dye to enhance contrast between different anatomical features.
________?
________?
_
4. Use the stage manipulator knobs to move the slide so the specimen is over the opening in the stage.
5. Look through the ocular lenses.
a. Adjust the ocular lenses toward and away from one another until you see just one image in a welldefined circle of light.
6. If your slide is positioned so that the image is in the center of the hole in the stage, but all you see is light,
it probably means the plane of focus is so far above or below your sample you can’t see it.
a. Use the coarse focus knob to slowly lower the stage until the image come into focus.
b. Use the fine focus knob to adjust it precisely.
7. Use the stage manipulator controls to center the specimen.
8. Use the fine focus knob to sharpen the image.
9. Rotate the 10X objective lens into place without changing anything and look through the oculars again.
However, you usually need to make slight adjustments in focus with fine focus knob only, as you go from
lower to higher power.
10. Each time you view an image, note that the image has been magnified by two “sets” of lenses. The objective
lens magnifies the image by 4X (low power), and the ocular lenses magnify that image 10X more. This means
that you are seeing the image 40 times its actual size. When you change the objective to 10x, what is the
total magnification of the image? ____________________________
11. Make sure that the image is in the very center of the field of view and the image is in sharp focus.
Without changing anything, rotate the 40X objective lens into place. Notice how close the tip of the lens is
to the cover slip. What is this distance called?
12. Bring the image into sharp focus with the fine focus knob only! If you use the coarse focus control when
the 40x objective is in place, the stage will probably elevate enough to smash the slide into the lens tip, and
potentially break the slide.
*If you adjust the fine focus, but still can’t see anything, return to using the 10X objective lens,
and reposition the slide. Then try the 40X objective lens again. Re-centering “lost” objects is a
very important skill.
Measuring field of view (field diameter) and specimens
Your teacher may want you to estimate the size of specimens you view with the microscope. For example how long
is a sperm tail? How big is a red blood cell? How thick is the stomach wall? Microscopes sometimes have a ruler
placed into one of the eyepiece lenses that makes these measurements easy, but ours do not have this feature.
We’ll use another strategy - a plastic ruler and a little math.
1.
Make sure the 4x objective is in viewing position. Place a clear plastic ruler with millimeter markings (1
cm=10mm) across the opening in the microscope stage. If the ruler is thin enough, you can gently slip it
under one side of the stage clip mechanism. Position the ruler so that one mark is at the very left edge of
the field of view.
2. Count the distance across the field from left to right. If the final millimeter is not complete, estimate the
portion of it in the field of view. For example, if you count 7 full millimeters, and about one-quarter of the
next one, record the field diameter as 7.25 mm. This is the distance across the entire field at 40x. If an
object covers the entire field like that shown below, the cell is 7.25 mm. If the object covers ½ the field,
how big is it?
Cell size ________
Cell size _________
***Record you’re the field diameter measurement for your microscope here: ______________. You
will need this throughout the quarter.
3. Estimate field diameters for the 10X and 40X lenses using the mathematical relationship below. Do not use
the ruler since it will damage the objectives and it is not precise enough.
FDA x A total mag = FDB x B total mag
Rearrange the equation to solve for the unknown:
(FDA x A total mag) / B total mag = FDB
FD 10X lens (mm) = (40/100) x measured field diameter with 4X lens (mm)
FD 40X lens (mm) = (40/400) x measured field diameter with 4X lens (mm)
***Record these field diameter values and on something that you will always bring to lab so that you can use
them in future exercises.****
40x total magnification: MEASURED
100x total magnification:_________________________
400x total magnification:_________________________
Using the Microscope: Examining Stained Specimens
Examine nervous tissue specimens indicated on the board
Draw what you see, note total magnification and field diameter so that you can make an estimate of
cell size
Make any notations that will help you remember what you saw