Objective:
To explain in short essays or
diagrams the process of diffusion and the movement of substances across the
cell membrane, at the level of 85% proficiency for each student.
In order to achieve this objective, you will need to be able
to:
- Explain Brownian motion and the effects of
temperature on Brownian motion.
- Explain differences in diffusion in gel and water.
- Determine the rate of diffusion of substances with
different molecular weights.
- Explain the movement of substances across an artificial
membrane.
- Explain the movement of substances across the plasma
membrane of blood cells placed in isotonic, hypertonic and hypotonic
solutions.
Materials:
Group Supplies
clean slide and coverslip
forceps
dropper bottle of whole milk
compound microscope
hot plate
10 microliter pipetters
petri dish containing agar gel
millimeter ruler
methylene blue concentrated solution
potassium permanganate concentrated solution
100 mL graduated cylinder
distilled water
0.9% sodium chloride solution (physiologic saline)
1.5% sodium chloride solution
gloves
lancets – spring loaded
alcohol and cotton
three
dialysis sacs*
small funnel
10 mL graduated cylinder
wax marker
three beakers (250 mL).
40% glucose solution
10% NaCl solution
dropper bottle of Benedict's solution
silver nitrate solution
test tube rack
3 test tubes
test tube holder.
Methods:
- Place a small drop of milk on a slide and cover carefully
with a coverslip. Allow the slide to stand on the microscope stage for
about 10 minutes before observing. While waiting, turn on the hot plate
and set to a low temperature.
- Observe the slide with high power. Keep the light as dim
as possible to increase the contrast.
- Place the slide you prepared on the warm hot plate for a few
seconds. Using forceps remove the slide and observe it under the
microscope again.
Results:
|
Temp
|
Movement of milk
|
|
cool
|
|
|
warm
|
|
Discussion:
- What can you conclude about the effect of increased
temperature on the kinetic energy of molecules?
- How has the rate of Brownian motion changed?
Methods:
- Avoid contact between your skin and the. Inject
10 microliters of potassium permanganate dye into the agar gel. Inject 10
microliters of methylene blue crystals into the agar approximately 10
centimeters away from the potassium permanganate crystal. Record the time.
- At 15-minute intervals, use the millimeter ruler to
measure the distance the dye has diffused from each crystal source. These
observations should be continued for 1 1/2 hours, and the results recorded
in the chart below.
- Compute the rate of the dye diffusion through the gel.
Results:
|
Time (min)
|
Diffusion of methylene
blue
(MW = 320)
|
Diffusion of
potassium permanganate (MW = 158)
|
|
|
(mm)
|
mm/min
|
(mm)
|
mm/min
|
|
15
|
|
|
|
|
|
30
|
|
|
|
|
|
45
|
|
|
|
|
|
60
|
|
|
|
|
|
75
|
|
|
|
|
|
90
|
|
|
|
|
Discussion:
- Which dye diffused more rapidly?
- What is the relationship between molecular weight and rate
of molecular movement (diffusion)?
- Why did the dye molecules move?
Methods:
- Place 10 micoliters of concentrated potassium permanganate
in a 100 mL graduated cylinder.
- Slowly as possible add distilled water so as not to
disturb the due and fill to the 100 mL mark.
Results:
Measure from the bottom of the graduated cylinder the number of millimeters
the dye has diffused.
Compute the rate of the dye's diffusion through water.
|
Time (min)
|
Diffusion of
potassium permanganate (MW = 158)
|
|
|
(mm)
|
mm/min
|
|
15
|
|
|
|
30
|
|
|
|
45
|
|
|
|
60
|
|
|
|
75
|
|
|
|
90
|
|
|
Discussion:
- Does the potassium permanganate dye move (diffuse) more
rapidly through water or the agar gel? Explain your answer.
Methods:
- Number threee beakers 1 to 3 with the wax marker, and fill
beakers #1 and 3 half full with distilled water, and fill beaker # 2 half
full with 40% glucose solution.
- Prepare the three dialysis sacs* one at a time. Using the
funnel, place 20 mL of the specified liquid in each (see Table below). Press out the air, fold over the open
end of the sac, and tie it securely. Before proceeding to the next sac,
quickly and carefully blot the sac dry by rolling it on a paper towel, and
weigh it. Record the weight, and then drop the sac into the beaker numbered
correspondingly (see Table below).
Be sure the sac is completely covered by the solution in the beaker,
adding more solution if necessary.
- Keep the sacs undisturbed in the beakers for 1 hour. (Use
this time to continue with other experiments).
- After an hour, quickly and gently blot all three sacs dry,
weigh and record weights. (see Table).
- Get a beaker of water boiling on the hot plate.
- Measure glucose and sodium chloride as described below:
- For Sac/Beaker #1:
Place 5 mL of Benedict's solution in each of two test tubes. Put 4 mL of
the beaker fluid into one test tube and 4 mL of the sac fluid into the
other. Mark the tubes for identification and then place them in a beaker
containing boiling water. Place in a boiling bath for 2 minutes. Remove
from boiling bath and allow cooling. If a green, yellow, or rusty red
precipitate forms, the test indicates the presence of glucose. If the
solution remains the original blue color, the test is negative for glucose.
- For Sac/Beaker #3:
Take a 5mL sample of the beaker fluid and put it in a clean test tube. Add
a drop of silver nitrate. The appearance of a white precipitate or
cloudiness indicates the presence of AgCl (silver chloride), and thus the
presence of sodium chloride.
*Dialysis sacs are selectively permeable
membranes with pores of a particular size. The selectivity of living membranes
depends on more than just pore size, but using the dialysis sacs will allow you
to examine selectivity due to this factor.
Results:
Table 1. Weight of Sacs Before and 1 hr After Submerging
|
Sac / Beaker solution
|
Weight before
|
Weight 1 hr after
|
|
Sac 1: 40% glucose
Beaker 1: distilled water
|
|
|
|
Sac 2: 40% glucose
Beaker 2: 40% glucose
|
|
|
|
Sac 3: 10% NaCl
Beaker 3: distilled water
|
|
|
Table 2. Reaction to Benedicts Solution and Silver Nitrate
|
Sac / Beaker
solution
|
Benedicts
|
Silver Nitrate
|
|
Sac #1: 40% glucose
|
|
XXX
|
|
Beaker #1: distilled water
|
|
XXX
|
|
Beaker #3: distilled water
|
XXX
|
|
Discussion:
- In which of the test situations did net osmosis occur?
- In which of the test situations did net dialysis occur?
- What conclusions can you make about the relative size of
glucose, NaCl, and water molecules?
- With what cell structure can the dialysis sac be compared?
Methods:
- Place a very small drop of 0.9% sodium chloride solution
(physiologic saline) on a slide. Clean your finger with a cotton ball and
isopropyl alcohol. Prick your finger and place a drop of blood to the
saline on the slide. Tilt the slide to mix, cover with a coverslip, and
immediately examine the preparation under the high-power lens. Place all
lancets and sharp materials in the “sharps container.” Place all other
materials that have been exposed to blood in the “biohazard bag.”
- Prepare another wet mount of blood, but this time use 1.5%
saline solution as the suspending medium. After 5 minutes, carefully
observe the red blood cells under high power.
- Using the slide from # 2 above, add a drop of distilled
water to the edge of the coverslip. Fold a piece of filter paper in half
and place its folded edge at the opposite edge of the coverslip; it will
absorb the saline solution and draw the distilled water across the cells.
Watch the red blood cells as they float across the field. After about 5
minutes have passed, describe the change in their appearance.
Results:
|
Bathing solution
|
Red blood cell appearance
|
|
0.9% sodium chloride solution (physiologic saline)
|
|
|
1.5% sodium chloride solution
|
|
|
distilled water
|
|
Discussion:
- Do the red blood cells retain their normal smooth
disc-like shape?
- What is happening to the normally smooth disc shape of the
red blood cells?
- How do your observations of test tube C in Experiment 1
correlate with what you have just observed under the microscope?