General Biology II – Major Life Processes in Plants and Animals
Membranes
adapted from ,Eberhard, C. 1990. Biological Membranes. In:General Biology Laboratory Manual. Saunders College Publishing. pp. 37-46
This lab activity is intended to provide an opportunity for students…
To observe evidence of some membrane transport mechanisms
To provide experience using the following membrane transport terms:
solute
solvent
concentration gradient
diffusion
osmosis
active transport
selectively permeable
osmotic pressure
isotonic
plasmolysis
hypotonic
turgor pressure
hypertonic
lysis
Objectives:
This lab aims to contribute to the development of the following components of the Cégep Champlain
St.Lawrence Science Program Graduate Profile:
I. Apply The Experimental Method
II. Take A Systematic Approach To Problem Solving
IV. Reason Logically
V. Communicate Effectively
VI. Learn In An Autonomous Manner
VII. Work As Members Of A Team
XI.Develop Attitudes Appropriate For Scientific Work
XII. Apply What They Have Learned To New Situations
This lab also contributes to the attainment of the following elements of the 00XU objective:
1.
2.
3.
To analyze the relationship between structure and function in multicelled organisms
To apply the concept of homeostasis to the study of systems in plants and animals.
To explain the function of conservation, regulation and reproduction in multicelled organisms.
Background Information:
The plasma membrane of a cell is not an
inert container; rather it is a dynamic barrier
that regulates the transport of whatever passes
into and out of the cell. Membranes also react to
external signal molecules, and the molecular
machinery for producing energy is located on
them.
In this laboratory exercise, you will
study two ways in which substances move in
and out of cells: diffusion, a passive physical
process that requires no energy input from the
cell, and active transport, in which the cell uses
energy to transport specific substances in
specific directions. In both cases, the molecules
move through the plasma membrane.
Membranes are lipid bilayers that are
hydrophobic barriers to all but a very few
hydrophilic molecules such as H2O. For
example, glucose, amino acids and charged ions
(e.g. Na+) can get across the membrane only by
passing through specific protein channels.
Small, hydrophobic molecules such as O 2, on the
other hand, readily pass through the lipid
bilayer membrane. Because certain molecules,
but not others can pass, the membrane is said
to be selectively permeable.
Large molecules and particles may enter
(endocytosis) or leave (exocytosis) the cell only if
they are enclosed in sacs called vesicles which
are formed by the membrane itself. These
Membranes
processes are fundamentally different from
diffusion and active transport because the
material does not pass through the membrane.
Small or hydrophobic molecules such as
O2, CO2, N2 and benzene pass freely through the
cell membrane by diffusion. As the diffusing
molecules spread out, they form a concentration
gradient. The diffusing molecules always move
down the concentration gradient from a higher
to lower concentration until they reach
equilibrium. Then they will be spread out
evenly and there will be no further net
movement. The free energy of the solution will
be lower, and the entropy (disorder) will be
higher after diffusion has occurred.
The diffusion of solute across a
membrane is called dialysis. Each solute will
move from the side where its concentration is
higher to the side with a lower concentration,
because the direction of movement is always
down the concentration gradient. Its movement
will be independent of the movement of the
other solutes. Most hydrophilic molecules
require specific carrier protein channels
(imbedded within the membrane) in order to
diffuse across the cell membrane; when a
protein carrier is involved, the process is known
as facilitated diffusion.
In the special case of diffusion where the
substance diffusing across the membrane is
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water, the process is called osmosis. During
osmosis, water will flow through protein
channels called aquaporins, from the side with
the most water and lower solute concentration,
to the side with the lower water and higher
solute concentration. Water will act like any
other diffusing molecule and will flow down its
concentration gradient.
The solution with the lower solute
concentration is hypotonic relative to the other
solution. Conversely, the more concentrated
solution is hypertonic relative to the first.
Osmotic pressure is a quantitative measure of
the force needed to oppose osmosis from pure
water into a solution. It is proportional to the
concentration of particles in the solution: the
hypotonic solution will have a lower osmotic
pressure than the hypertonic one.
Cells cannot control the process of
osmosis directly since osmosis is a spontaneous
downhill process requiring no energy input.
Cells can and must control their water content,
however, in order to keep their solutes at the
correct concentrations required for life
processes. One strategy is to remove excess
water with special structures such as the
contractile vacuoles of some protists. Another is
to increase their solute concentration by
pumping in ions, thereby permitting water to
enter the cell by osmosis. If a cell has a cell wall,
the mechanical strength of the wall will exert
turgor pressure against the cell membrane to
prevent the influx of water by osmosis. A plant
keeps from wilting because the tonoplast
membrane controls the solute concentration
inside the central vacuole so that turgor is
maintained. If there is no cell wall, excess water
entering will cause the cell to swell and undergo
lysis (bursting). Cells exposed to hypertonic salt
solutions will shrink unless they can raise their
own solute concentration somehow. If there is a
cell wall, the cytoplasm pulls away in the
process of plasmolysis. Many animal cells have
no protection against shrinking or lysis and can
exist only in isotonic solutions.
Many substances are brought into the
cell or removed from the cell against their
concentration gradients in the process of active
transport. As in facilitated diffusion, the process
of active transport is highly specific in that each
particular substance to be carried across the
membrane must be transported by a specific
protein carrier imbedded within the membrane.
These membrane proteins require ATP energy
supplied by cellular respiration. Thus active
transport is coupled with respiration. If there is
no cell respiration, or if the integrity of the
membrane is destroyed, active transport cannot
occur.
B
Procedures
20 mm
I. Diffusion in a Solid
A
1. Obtain a Petri dish of 1.5% agar and turn it upside down. Use a
waterproof marker to make a dot in the center of the Petri dish and label it
A. Use a ruler to measure 20 mm from A and make a second dot, B. Make
a third dot, C 20 mm from A and as far away dot B as possible.
20 mm
C
2. Use a small piece of a straw attached to a pipette dropper bulb to make holes in the agar at
points A, B, and C. Pinch the dropper bulb, press the straw into the agar, release the bulb and
remove the straw. If you are lucky, the segment of agar will remain the in straw.
3. Add the following solutions to the appropriate holes. DO NOT over fill the holes and try to fill
them all to about the same level. Use caution when handling the solutions as some are toxic.
A: 1M AgNO3
silver nitrate
B: 1M NaCl
sodium chloride
C: 1M K3Fe(CN)6 potassium ferricyanide. (TOXIC! handle with care)
4. Place the Petri dish in a dark area. After about an hour you should be able to see colored bands
forming where solutions B and C meet solution A (silver nitrate)
5. When the incubation period is over (record results on Results worksheet)
a. record the colors of the bands that appear
b. measure the distance between the edge of each hole and the colored band.
c. calculate the ratio of the distance moved of the different ions
6. Discard the Petri dish when you have completed your measurements.
Membranes
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II. Osmosis in a Living Cell
1. Add a small amount of water to a microscope slide, place one Elodea leaf in the water drop and
apply a coverslip.
2. Examine the leaf under high power (40X lens, 400X magnification). Make a simple sketch of a
SINGLE cell.
3. Add a drop or two of 10% NaCl solution to one edge of the coverslip. Avoid spilling NaCl on the
microscope stage, condensor or lenses since salt can scratch these surfaces.
4. While looking through the microscope, touch the edge of a Kimwipe to the opposite side of the
coverslip to pull the salt solution through and observe the Elodea cells in a hypertonic
environment. Briefly sketch a single cell, emphasizing the change.
5. Add a few drops of distilled water to the edge of the coverslip and pull it through with a
Kimwipe as before while watching the cell. Observe changes to the cell.
6. When you have completed your observations, discard the microscope slide in the glass waste
bucket.
III. Osmosis in a Model Cell
1. Obtain a short piece of dialysis tubing (12 – 15 cm) that has been softened by soaking in
distilled water. Fold one end over, and tie it tightly with a piece of thin string or strong thread.
2. Fill the bag with Solution A, a liquid "meal" containing:
25.0% glucose
0.5% egg albumin
1.0% starch
3. Squeeze out all the air, fold over the top and tie tightly. The bag should be limp. Rinse with tap
water.
4. Place the limp bag in a small beaker and add enough water to cover the bag. Add I2KI to the
water until the solution becomes slightly yellow (about 4 - 5 drops). Stir gently to disperse the
iodine and leave the beaker undisturbed for about an hour.
-When the incubation period has ended, perform the following procedures
5. Observe and record changes to the dialysis bag. (size, shape, color, etc.)
Test for the presence of sugars in the surrounding water.
6. Prepare three test tubes.
a. positive control: 1 mL Benedict’s solution + 0.5 mL of sugar solution
b. negative control: 1 mL Benedict’s solution + 0.5 mL of distilled water
c. sample: 1 mL of Benedict's solution + 0.5 mL of iodine water from your beaker
7. Place the three test tubes in a boiling water bath for 2 minutes. Compare the results of the
sample to the positive and negative controls. Record your results on the Results worksheet.
-if solution remains clear – no sugars present
-if the solution becomes cloudy due to the formation of a white precipitate
(copper oxide) this indicates that reducing sugars are present in the water.
Test for the presence of proteins in the surrounding water.
8. Prepare three test tubes.
a. positive control: 1 mL Biuret reagent + 1 mL of protein solution
b. negative control: 1 mL Biuret reagent + 1 mL of distilled water
c. sample: 1 mL of Biuret reagent + 1 mL of the iodine water from your beaker
9. Observe the color of the solutions. Compare the results of the sample to the positive and
negative controls. Record your results on the Results worksheet.
-if solution remains blue – no protein present
-if the solution becomes purple, this indicates that proteins are present.
Membranes
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IV. Active Transport
1. Measure two, 2 g samples of yeast and dissolve the yeast in warm water. You may do this
directly in the weigh boat.
2. Add one of the yeast samples to a 250 mL Erlenmeyer flask designated Flask “A”. Add 25 mL of
0.75% Na2CO3 (sodium carbonate) and mix well.
3. Gently boil the contents of Flask A for 2 minutes and allow to cool. The cells are now dead.
4. Add 25 mL of 0.02% neutral red. Record the color of suspension “A” in the Results worksheet.
(initial A)
5. To Flask “B”, add 25 mL of 0.75% Na2CO3 and 25 mL of 0.02% neutral red. Record the color of
suspension “B” in the Results worksheet. (initial B)
6. Add the remaining yeast sample to Flask “B”, swirl the flask and observe a change in color.
7. After a few minutes record the colors of the contents of both flasks. (final A and B)
Either the cells in Flask “B” put out something to change the solution or the dye entered the cells and
was changed in the cytoplasm. To test this, compare cells in Flasks A and B:
8. Filter a portion of the contents of Flask A into a test tube. Record the color of the yeast cells (on
the filter paper) and the color of the solution (in the test tube).
9. Filter a portion of the contents of Flask B into a test tube. Record the color of the yeast cells (on
the filter paper) and the color of the solution (in the test tube).
10. Add an equal volume of 0.75% acetic acid to the remaining contents of Flask A to acidify the
suspension. Record the color of the suspension in Flask A.
11. Filter a portion of this suspension. Record the color of the cells.
Notes for Active Transport experiment
yellow color only occurs when neutral red is mixed with Na 2CO3
if cells are yellow, then neutral red and Na2CO3 are both inside the cells
if cells are red, then neutral red is inside the cells (or neutral red plus acetic acid)
if cells are unchanged in color, then neither neutral red or Na 2CO3 are inside the cells
Rinse all glassware used, discard used filter papers, and clean up your work area.
Sample Marking Grid
/7 Diffusion in a Solid
/1 results described in single sentence
/1 Table 1a
/1 Table 1b
/1 Table 1c
/3 explanation of colored band and of diffusion rate
why did bands form?
why did bands form at different distances?
justified with observations and diffusion principles
/10 Osmosis in a Model Cell
/2 Table 2 results
/2 did water move across membrane? yes/no
evidence based on observations
if yes, in which direction? state evidence
/2 did glucose move across membrane? yes/no
evidence based on observations
if yes, in which direction? state evidence
/2 did egg albumin move across membrane? yes/no
evidence based on observations
if yes, in which direction? state evidence
/2 did starch move across membrane? yes/no
evidence based on observations
if yes, in which direction? state evidence
/7 Osmosis in a Living Cell
/1 Figure 1
/1 Figure 2
Membranes
/2
/3
description of results (present; clear; thorough)
explanation
logical
justified with observations
justified with membrane transport principles
/10 Active Transport
/2 Table 3
/3 summary of Flask A – dead cells
are the cells in flask A permeable to dye?
yes/no
evidence/observation
are the cells in flask A permeable to Na2CO3?
yes/no
evidence/observation
are the cells in flask A permeable to HOAc?
yes/no
evidence/observation
/2 summary of Flask B –living cells
are the cells in flask B permeable to dye?
yes/no
evidence/observation
are the cells in flask B permeable to Na2CO3?
yes/no
evidence/observation
/3 explanation
logical
justified with observations
justified with membrane transport principles
/34 Total
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